1. Ukuqonda kokuqala

Kulesi sigaba, sidinga ukuqonda imiqondo ethile namagama, ukuze sigweme ukwenza amaphutha phambi kwabadala bethu, njengokuthi:

Q: Uyini umehluko phakathi kwe-RT-PCR, qPCR, Real-time PCR, kanye ne-RT-PCR yesikhathi sangempela?

Impendulo: I-RT-PCR iwukuhlanekezela okulotshiweyo kwe-PCR(reverse transcription PCR, RT-PCR), okuwuhlobo olusetshenziswa kakhulu lwe-polymerase chain reaction (PCR).Ku-RT-PCR, umucu we-RNA uhlehliswa uguqulelwe ku-DNA ehambisanayo, ebese isetshenziswa njengesifanekiso sokukhulisa i-DNA yi-PCR.
I-Real-time-PCR ne-qPCR(I-Quantitative Real-ltime-PCR) iyinto efanayo, zombili ziyi-PCR yobuningi besikhathi sangempela, okusho ukuthi umjikelezo ngamunye we-PCR unamarekhodi edatha yesikhathi sangempela, ngakho inani lezifanekiso zokuqala zingalungiswa ukuhlaziywa okunembile.

Nakuba kokubili i-Real-time PCR (real-time fluorescent quantitative PCR) kanye ne-Reverse transcription PCR (reverse transcription PCR) kubonakala kufushanisiwe njenge-RT-PCR, umhlangano wamazwe ngamazwe uthi: I-RT-PCR iqondise ngokuqondile ekulotshweni okuhlanekezelwe.I-PCR , I-PCR yesikhathi sangempela ivamise ukufushaniswa njenge-qPCR (ubuningi be-PCR yesikhathi sangempela).

Futhi i-RT-PCR (RT-qPCR) yesikhathi sangempela, I-PCR ebhalwe emuva ehlanganiswe nobuchwepheshe bokulinganisa be-fluorescent.: qala uthole i-cDNA (RT) kusukela ekulotshweni okuhlanekezela kwe-RNA, bese usebenzisa i-Real-time PCR ukuze uthole ukuhlaziywa komthamo (qPCR).Amalabhorethri amaningi enza i-RT-qPCR, okusho ukuthi, ucwaningo nge-RNA expression down-regulation, ngakho-ke i-qPCR wonke umuntu akhuluma ngayo elabhorethri empeleni ibhekisele ku-RT-qPCR, kodwa ungakhohlwa ukuthi kusekuningi ukuhlolwa kwe-DNA ezinsizeni zomtholampilo.Ukuhlaziywa komthamo, njengokutholwa kwegciwane le-hepatitis B HBV.

Umbuzo: Ngemva kokufunda i-PCR yobuningi be-fluorescent, kungani kufanele ucezu olukhulisiwe lulawulwe phakathi kwebanga elingu-80-300bp?

Phendula: Ubude bokulandelana kofuzo ngalunye buhlukile, amanye ama-kb amaningana, amanye amakhulu e-bp, kodwa sidinga kuphela ukuthi ubude bomkhiqizo bube ngu-80-300bp uma siklama ama-primer, amafushane kakhulu noma amade kakhulu awafanele ukutholwa kwe-PCR yobuningi be-fluorescent.Ucezu lomkhiqizo lufushane kakhulu ukuthi lungahlukaniswa ku-primer-dimer.Ubude be-primer-dimer cishe bungama-30-40bp, futhi kunzima ukuhlukanisa ukuthi i-primer-dimer noma umkhiqizo uma ingaphansi kuka-80bp.Uma ucezu lomkhiqizo lude kakhulu, ludlula u-300bp, luzoholela kalula ekusebenzeni kahle kwe-amplification futhi alukwazi ukubona kahle inani lofuzo.

Isibonelo, uma ubala ukuthi bangaki abantu ekilasini, udinga kuphela ukubala ukuthi mingaki imilomo.Okufanayo kuyiqiniso uma uthola izakhi zofuzo, udinga kuphela ukuthola ukulandelana okuthile kofuzo ukumela Konke ukulandelana okuzokwenza.Uma ufuna ukubala abantu, udinga ukubala kokubili imilomo namakhala, izindlebe, nezibuko, futhi kulula ukwenza amaphutha.

Ukwandisa, ocwaningweni lwebhayoloji, kunamacala amaningi ocwaningo ukusuka endaweni kuya endaweni, ngenxa yokuthi ukulandelana kwezakhi zofuzo kwanoma yiluphi uhlobo lude kakhulu, akudingekile futhi akwenzeki ukukala zonke izingcezu, njengokulandelana kwebhaktheriya 16S, okuwukwenza ukulandelana kokugcina kwe-bacteria Assays ukuze kutholakale inani labantu abathile bamagciwane.

Q: Yibuphi ubude obufanele bomklamo wokuqala we-qPCR?

Phendula: Ngokuvamile, ubude be-primer bumayelana ne-20-24bp, okungcono.Vele, kufanele sinake inani le-TM le-primer lapho siklama i-primer, ngoba lokhu kuhlobene nezinga lokushisa elilungile lokudonsa.Ngemuva kokuhlolwa okuningi, kufakazelwe ukuthi i-60°C iyivelu ye-TM engcono.Uma izinga lokushisa le-anneal liphansi kakhulu, lizoholela kalula ekukhuliseni okungaqondile.Uma izinga lokushisa le-anneal liphezulu kakhulu, ukusebenza kahle kokukhulisa kuzoba phansi uma kuqhathaniswa, ukuphakama kwejika lokukhulisa kuzoqala kamuva, futhi inani le-CT lizobambezeleka.

Umbuzo: Ihluke kanjani indlela yokudayi kunendlela yokuhlola?

Impendulo: Indlela yokudayaAbanye odayi be-fluorescent, njenge-SYBR Green Ⅰ, i-PicoGreen, i-BEBO, njll., abakhiphi ukukhanya ngokwabo, kodwa bazokhipha i-fluorescence ngemva kokubophezela ku-groove encane ye-DNA enezintambo ezimbili.Ngakho-ke, ekuqaleni kokusabela kwe-PCR, umshini awukwazi ukubona isignali ye-fluorescent.Lapho ukusabela kufinyelela esigabeni sokunweba, umucu ophindwe kabili uyavuleka, bese umucu omusha uhlanganiswa ngaphansi kwesenzo se-DNA polymerase, bese i-molecule ye-fluorescent ibophezela ku-dsDNA encane groove.Njengoba inani lemijikelezo ye-PCR likhula, odayi abaningi ngokwengeziwe bahlanganiswa ne-DNA enemicu ephindwe kabili, futhi isignali ye-fluorescent nayo ithuthukiswa ngokuqhubekayo.Indlela yokudaya isetshenziswa kakhulu ocwaningweni lwesayensi.
I-PS: Qaphela lapho wenza ucwaningo, udayi kufanele uhlanganiswe ne-DNA yomuntu, qaphela ukuwuguqula ube umuntu okhanyayo.

Indlela yokudaya (kwesokunxele) Indlela yokuphenya (kwesokudla)
I-PS: Qaphela lapho wenza ucwaningo, udayi kufanele uhlanganiswe ne-DNA yomuntu, qaphela ukuwuguqula ube umuntu okhanyayo.

I-SYBR Green Ⅰ ibophezela emseleni omncane we-DNA

Indlela yokuphenyaI-Taqman probe iwuphenyo oluvame ukusetshenziswa kakhulu lwe-hydrolysis.Kuneqembu le-fluorescent ekugcineni kuka-5′ wophenyo, ngokuvamile i-FAM, futhi uphenyo ngokwalo luwuchungechunge oluhambisanayo nofuzo oluqondiwe.Kukhona iqembu le-fluorescent quenching ekugcineni kuka-3′.Ngokomgomo we-fluorescence resonance energy transfer (Förster resonance energy transfer, FRET), lapho iqembu lentatheli yefluorescent (donor fluorescent molecule) kanye neqembu le-fluorescent elicishayo (i-acceptor fluorescent molecule) bayajabula Lapho i-spectra igqagqene nebanga liseduze kakhulu (7-10 excitation of the donor molecule) yamukela i-molecule ye-fluorescent , kuyilapho i-autofluorescence ibuthakathaka.Ngakho-ke, ekuqaleni kokusabela kwe-PCR, lapho uphenyo lukhululekile futhi luqinile ohlelweni, iqembu lentatheli ye-fluorescent ngeke likhiphe i-fluorescence.Lapho i-anneal, i-primer kanye ne-probe kubophezela kusifanekiso.Phakathi nesigaba sokunwetshwa, i-polymerase ihlanganisa ngokuqhubekayo amaketanga amasha.I-DNA polymerase inomsebenzi we-5'-3′ we-exonuclease.Lapho ifinyelela kuphenyi, i-DNA polymerase izosebenzisa i-hydrolyze uphenyo kusukela kusifanekiso, ihlukanise iqembu lentatheli ye-fluorescent eqenjini le-quencher fluorescent, futhi ikhulule isignali ye-fluorescent.Njengoba kunobudlelwano obubodwa phakathi kwe-probe kanye nesifanekiso, indlela yokuhlola iphakeme kunendlela yokudaya ngokunemba nokuzwela kokuhlolwa.Indlela ye-probe isetshenziswa kakhulu ekuxilongeni.

Q: Kuyini i-absolute quantification?Iyini i-Relative Quantification?

Phendula: Ukulinganisa okuphelele kubhekisela ekubalweni kwenombolo yekhophi yokuqala yesampula ezohlolwa yi-qPCR, njengokuthi mangaki amagciwane e-HBV aku-1ml yegazi.Umphumela otholwe ngobuningi obuhlobene uwushintsho lwenani lofuzo oluqondiwe kusampula ethile ehlobene nenye isampula yereferensi, futhi isisho sofuzo silawulwa phezulu noma siqondiswa phansi.

Q: Ingabe inani lokukhishwa kwe-RNA, ukuhlehlisa ukusebenza kahle kokuloba, kanye nempumelelo yokukhulisa i-amplification kuzothinta imiphumela yokuhlolwa?
Umbuzo: Ingabe isampuli yokulondoloza, izinza, izinza, ama-reagents okulotshwa ahlehlayo, nezinto ezisebenzisekayo zokudlulisa ukukhanya kuzothinta imiphumela yokuhlolwa?
Q: Iyiphi indlela engalungisa idatha yokuhlola?

Mayelana nalezi zinkinga, sizozichaza ngokuningiliziwe ezigabeni ezithuthukisiwe nezithuthukile ngezansi.
2. Ulwazi oluphambili

Mayelana ne-PCR yesikhathi sangempela ye-fluorescent quantitative, kufanele siqaphele iqiniso lokuthi izinkulungwane zamaphepha ocwaningo lwesayensi ashicilelwa njalo ngonyaka, phakathi kwawo ubuchwepheshe be-PCR bomthamo we-fluorescent akuyona inombolo encane.

Uma lingekho izinga elijwayelekile lokulinganisa isilingo se-PCR somthamo we-fluorescent, imiphumela ingase yehluke kakhulu.Kufuzo olufanayo lohlobo olufanayo, olunendlela yokucubungula efanayo, imiphumela yokuthola nayo izohluka kakhulu, futhi kuzoba nzima kwabafika sekwephuzile ukuphinda imiphumela efanayo.Wena Akekho owaziyo ukuthi yikuphi okulungile nokungalungile.

Ingabe lokhu kusho ukuthi i-fluorescent quantitative PCR iwubuchwepheshe bokukopela noma ubuchwepheshe obungathembeki?Cha, kungenxa yokuthi i-fluorescent quantitative PCR izwela kakhulu futhi inembe kakhulu, futhi ukusebenza okungalungile okuncane kuzoveza imiphumela ehluke ngokuphelele.Ukulahlekelwa okuncane kuqhele ngamakhilomitha ayinkulungwane.Umbhali we-athikili angase ahlukunyezwe ngokuphindaphindiwe ababuyekezi.Ngesikhathi esifanayo, ababuyekezi bejenali nabo kunzima ukukhetha emiphumeleni yokuhlola ehlukene.

Sekukonke, kukhomba ukuntula ukuvumelana ekuhlolweni kwe-PCR yesikhathi sangempela.Kuze kube manje, ososayensi abakhulu embonini baqala ukwenza amazinga,idinga abanikeli ukuthi banikeze eminye imininingwane edingekayo yokuhlolwa neyokucubungula idatha (okuhlanganisa idatha edingekayo) esihlokweni ukuze kuhlangatshezwane nalawa mazinga.

Ababuyekezi bangahlulela ikhwalithi yokuhlolwa ngokufunda le mininingwane;abafundi besikhathi esizayo bangaphinda basebenzise lokhu ukuze baphinde ukuhlola noma bathuthukise isilingo.Bese imiphumela yokuhlolwa etholwe ngale ndlela igcwele ulwazi, ikhwalithi ephezulu, futhi iyasebenziseka.

I-MIBBI (Ulwazi Oluncane Lophenyo Lwebhayoloji kanye Nezokwelapha -http://www.mibbi.org) kwavela.I-MIBBI iphrojekthi ehlinzeka ngamazinga okuhlolwa.Ishicilelwe ngokwemvelo.Le phrojekthi ihloselwe ukuhlola okuhlukahlukene kwebhayoloji, okuhlanganisa i-cell biology, i-Microarray, i-qPCR esizoxoxa ngayo manje, njll., futhi inikeza uhlobo ngalunye lokuhlola lapho kuhanjiswa imibhalo yesandla.Lolo lwazi kufanele luhlinzekwe ngaso sonke isikhathi.

Kuphrojekthi ye-MIBBI, kunezihloko ezimbili ezihlobene ne-fluorescent quantitative PCR, okungukuthi:
·I-RDML (Ulimi Lwemakhaphu Yedatha Yesikhathi Sangempela) – ulimi oluhlelekile nomhlahlandlela wokubika wedatha ye-PCR yesikhathi sangempela;
·MIQE (Ulwazi Oluncane Lokushicilelwa Kokuhlolwa Kwesikhathi Sangempela Kwe-PCR) – ubuncane bemininingwane yokushicilela ama-athikili mayelana nokuhlolwa komthamo we-PCR wesikhathi sangempela.
Okokuqala, ake sikhulume nge-RDML, ukucaciswa kwamagama.

Uma kungekho ncazelo ejwayelekile yazo zonke izinto, akunakwenzeka ukuqhubeka nengxoxo, yingakho incazelo yemigomo ibaluleke kakhulu ekuhlolweni.
Amagama asetshenziswa ocwaningweni lobuningi be-fluorescent PCR afaka phakathi okuqukethwe okulandelayo.U-QIAGEN usenzele isifinyezo esihle kakhulu.Okulandelayo komile konkeizimpahla .

Ijika lokukhulisa
Ijika le-amplification lisho ijika elenziwe phakathi nenqubo ye-PCR, nenombolo yomjikelezo njenge-abscissa kanye nokuqina kwe-fluorescence yesikhathi sangempela phakathi nokusabela njengokuhleleka.

Ijika elihle kakhulu lokukhulisa i-amplification kufanele libe nezici ezilandelayo: isisekelo siyisicaba noma sehle kancane, futhi akukho mkhuba osobala obheke phezulu;iphoyinti le-inflection lejika licacile, futhi umthambeka wesigaba se-exponential ulingana nokusebenza kahle kokukhulisa.Uma umthambeka umkhulu, kulapho kukhuphuka khona ukusebenza kahle kokukhulisa;ijika le-amplification jikelele Ukuhambisana kuhle, okubonisa ukuthi ukusebenza kahle kokukhulisa ishubhu ngayinye kuyafana;isigaba se-exponential sejika le-amplification lamasampuli okugxila okuphansi sisobala.

Isisekelo (Isisekelo)
Isisekelo izinga lomsindo lomjikelezo wokuqala, ngokuvamile kulinganiswa phakathi komjikelezo wesi-3 nowe-15, ngenxa yokuthi ukukhuphuka kwenani le-fluorescence okubangelwa umkhiqizo wokukhulisa amandla akukwazi ukutholwa phakathi nalesi sikhathi.Inombolo yemijikelezo esetshenziselwa ukubala isisekelo ingahlukahluka futhi ingase idinge ukuncishiswa uma amanani aphezulu esifanekiso asetshenziswa noma uma izinga lenkulumo yofuzo oluqondiwe liphezulu.

Ukusetha isisekelo kudinga ukubuka idatha ye-fluorescence kusukela ku-linearity amplification curve .Isisekelo sisethwe ukuze ukukhula kwejika lokukhuliswa kuqale ngenombolo yomjikelezo enkulu kunenombolo ephezulu yomjikelezo wesisekelo.Imigqa eyisisekelo idinga ukusethwa ngayinye ngokulandelana kwethagethi ngayinye.Amanani e-fluorescence amaphakathi atholwe emijikelezweni yokuqala adinga ukukhishwa kumanani e-fluorescence atholakala emikhiqizweni ekhulisiwe.Izinguqulo zakamuva zesofthiwe ehlukahlukene ye-Real-Time PCR ivumela ukulungiselelwa okuzenzakalelayo kwezilungiselelo eziyisisekelo zamasampuli ngamanye.

Phakathi nemijikelezo embalwa yokuqala yokusabela kokukhulisa i-PCR, isignali ye-fluorescence ayishintshi kakhulu.Ukusondela emgqeni oqondile kubizwa ngokuthi isisekelo, kodwa uma sibhekisisa imijikelezo embalwa yokuqala, sibona ukuthi ngaphakathi kwesisekelo kukhona okwenzekayo esithombeni esingezansi.

Ingemuva libhekise ku
inani le-fluorescence elingaqondile ekuphenduleni .Isibonelo: ukucisha i-fluorescence engasebenzi kahle;noma inombolo enkulu yezifanekiso ze-DNA ezinemiphetho ekabili ngenxa yokusetshenziswa kwe-SYBR Green.Izingxenye zangemuva zesiginali zisuswa ngokwezibalo i-algorithm yesofthiwe ye-PCR Yesikhathi Sangempela.

Isiginali yentatheli
Isignali yentatheli ibhekisela kusignali ye-fluorescent ekhiqizwe i-SYBR Green noma ama-fluorescent anelebuli yokulandelana okuqondile ngesikhathi se-PCR Yesikhathi Sangempela.

Isiginali Yentatheli Ejwayelekile (RN)
I-RN ibhekisela kubukhulu be-fluorescence bedayi yentatheli ehlukaniswa amandla e-fluorescence we-passive reference idayi elinganiswa kumjikelezo ngamunye.

I-Passive Reference Daye
Kwamanye ama-PCR esikhathi sangempela,udayi we-fluorescent i-ROX isetshenziswa njengereferensi yangaphakathi yokwenza isignali ye-fluorescent ijwayelekile.Ilungisa ukuhlukahluka ngenxa yepayipi elingalungile, indawo ekahle, kanye nokuguquguquka kwe-fluorescence ngendlela efanele.

Umkhawulo we-fluorescence (i-threshold)
yalungiswa ngaphezu kwevelu yangemuva futhi yangaphansi kakhulu kwevelu ye-plateau yejiko lokukhulisa.Kumelwe ilele endaweni ewumugqa wejika lokukhulisa, imele uhla lwelogi lomugqa wokutholwa kwe-PCR.Ama-Threshold kufanele amiswe ekubukeni kwejika lokukhula kwelogi ukuze isigaba somugqa welogi se-PCR sibonakale kalula.Uma kunezinhlobo eziningi zofuzo eziqondiwe ku-Real-Time PCR, umkhawulo kufanele usethelwe ithagethi ngayinye .Ngokuvamile, isignali ye-fluorescence yemijikelezo yokuqala engu-15 yokusabela kwe-PCR isetshenziswa njengesignali yangemuva ye-fluorescence, futhi umkhawulo we-fluorescence uphindwe izikhathi ezingu-10 ukuchezuka okujwayelekile kwesignali ye-fluorescence yemijikelezo yokuqala engu-3 kuya kweyi-15 ye-PCR, futhi umkhawulo we-fluorescence usethwe ku-amponplential ye-PC.Ngokuvamile, ithuluzi ngalinye linomkhawulo walo we-fluorescence osethiwe ngaphambi kokusetshenziswa.

I-Cycle Threshold (CT) noma i-Crossing Point (CP)
Umjikelezo lapho ijika lokukhulisa liwela khona umkhawulo (okungukuthi, iphuzu lapho ukutholwa kwe-fluorescence kukhula kakhulu).I-CT ingaba ingxenyenanyana futhi nenani lesifanekiso sokuqala singabalwa.Inani le-CT limelela inani lemijikelezo etholwayo lapho isignali ye-fluorescent kushubhu yokusabela ye-PCR ngayinye ifinyelela embundwini omisiwe.Kunobudlelwano bomugqa phakathi kwevelu ye-CT yesifanekiso ngasinye kanye ne-logarithm yenombolo yokuqala yekhophi yesifanekiso,ephakeme inombolo yekhophi yokuqala, i-CT value encane, futhi ngokuphambene nalokho.Ijika elijwayelekile lingenziwa ngokusebenzisa indinganiso enenombolo yekhophi yokuqala eyaziwayo, lapho i-abscissa imele inani le-CT, futhi i-ordinate imele i-logarithm yenombolo yokuqala yekhophi.Ngakho-ke, inqobo nje uma inani le-CT lesampula elingaziwa litholwa, inombolo yokuqala yekhophi yesampula ingabalwa kusukela kujika elijwayelekile.

ΔCT inani
ΔCT inani liyachazaumehluko phakathi kofuzo oluqondiwe kanye nenani le-CT lereferensi elihambisanayo elihambisanayo, njengofuzo lokugcina indlu, futhi isetshenziselwa ukwenza ngokwejwayelekile inani lesifanekiso esisetshenzisiwe:
⇒ΔCT = CT (ufuzo oluqondiwe) - CT (ufuzo lwereferensi olungapheli)

ΔΔCT Inani
Inani le-ΔΔCT lichaza umehluko phakathi kwevelu ye-ΔΔCT yencazelo yesampula yentshisakalo (isb, amaseli avuselelwe) kanye nenani elisho ΔΔCT lesampula yereferensi (isb, amaseli angavuselelwe).Isampula yereferensi ibizwa nangokuthi isampula yokulinganisa futhi wonke amanye amasampuli ajwayelekile kulokhu ukuze kulinganiswe inani elihlobene:
⇒ΔΔCT = isilinganiso ΔCT (isampula yentshisekelo) - isilinganiso ΔCT (isampula yereferensi)

Izinhlobo zereferensi ze-Endogenous (izinhlobo zereferensi ezingapheli)
Amaleveli wenkulumo wezakhi zofuzo zereferensi engapheli, njengezakhi zofuzo zokugcina indlu (izakhi zofuzo zokunakekela indlu), awahlukani phakathi kwamasampuli.Ukuqhathanisa amanani e-CT ofuzo lofuzo oluqondiswe kufuzo lofuzo oluqondiwe kuvumela izinga lenkulumo yofuzo oluqondiwe ukuthi lenziwe libe elijwayelekile libe inani lokufakwayo kwe-RNA noma i-cDNA (bona isigaba samavelu e-ΔCT ngenhla).

Izakhi zofuzo zesithenjwa zangaphakathi zilungileukucekelwa phansi kwe-RNA okungenzeka noma ukuba khona kwama-enzyme inhibitor kumasampuli e-RNA, kanye nokuhluka kokuqukethwe kwe-RNA, ukusebenza kahle kokuhlehla kokulotshwayo, ukutholwa kwe-nucleic acid, nokuphathwa kwesampula.Ukuze sikhethe ufuzo olulungile lwereferensi, silungise i-algorithm ukuze sivumele ukukhethwa kwayo kwereferensi eningi encike kusilungiselelo sokuhlola.

Ukulawula kwangaphakathi
Ukulandelana kokulawula okukhuliswa ngokusabela okufanayo nokulandelana okuqondiwe futhi kuphenywe nge-probe ehlukile (okungukuthi, ukwenza i-duplex PCR).Izilawuli zangaphakathi zivame ukusetshenziselwa ukukhipha ukukhulisa okuhlulekile, njengalapho ukulandelana okuqondiwe kungatholwa.
Isampula Yokulinganisa
Isampula yereferensi (isibonelo, i-RNA ehlanziwe kusukela kumugqa weseli noma izicubu) esetshenziswa ekubalweni okuhlobene ukuze kuqhathaniswe wonke amanye amasampuli ukuze kutholwe ileveli yesisho esihlobene sofuzo.Isampuli yokulinganisa ingaba yinoma iyiphi isampuli, kodwa ngokuvamile iwukulawula (isibonelo, isampula engalashwanga noma isampula kusukela kusikhathi uziro wokuhlolwa).

Izilawuli ezinhle
sebenzisa ukulawula ukusabela ngeamanani aziwayo esifanekiso.Izilawuli ezinhle zivame ukusetshenziselwa ukuhlola ukuthi isethi yokuqala noma isethi ye-primer–probe isebenza kahle nokuthi ukusabela kumiswe ngendlela efanele.

Akukho Ukulawula Isifanekiso (NTC)
Ukusabela kokulawula okuqukethe zonke izingxenye ezidingekayo zokusabela kokukhulisa ngaphandle kwesifanekiso, esivame ukushintshwa ngamanzi.Ukusetshenziswa kwe-NTC kungathola ukungcola okubangelwa ukungcoliswa kwe-reagent noma i-DNA yangaphandle, ngaleyo ndlela kuqinisekiswe ubuqiniso nokuthembeka kwedatha yokuthola.Ukwenyuswa kokulawula kwe-NTC kukhombisa ukungcola.

Akukho ukulawula kwe-RT (NRT)
Inqubo yokukhipha i-RNA ingase iqukathe i-DNA eyinsalela ye-genomic, eyingozi kakhulu futhi eyimbangela ethinta ikhwalithi yedatha nesitha esingokwemvelo se-qPCR, ngakho-ke lapho uklama ukuhlola, kufanele yakhelwe ukukhulisa ukutholwa kwe-RNA kuphela.Kunezindlela ezimbili, eyodwa ukuklama ama-primer kuwo wonke ama-intron, enye iwukukhipha ngokuphelele i-DNA, iyiphi engcono, okuzoxoxwa ngayo kamuva.Isilawuli se-NTR yisibuko somlingo sokubona ukungcola kwe-DNA.Uma kukhona ukukhuliswa, kusho ukuthi kukhona ukungcola.

Amazinga
Amazinga amasampula okugxiliswa okwaziwayo noma inombolo yokukopisha asetshenziselwa ukwakha ijika elijwayelekile.Ukuze kuqinisekiswe ukuzinza kwendinganiso, ucezu lofuzo luvamise ukuhlanganiswa ku-plasmid futhi lusetshenziswe njengendinganiso.

Ijika elijwayelekile
ngokuvamile ihlanjululwa okungenani ibe ama-concentration gradients angu-5 ngomkhiqizo ojwayelekile ngokwesilinganiso esiphindeka kabili, futhi amaphuzu angu-5 adwetshwa kuzixhumanisi yenani le-CT nenombolo yokukopisha, futhi amaphuzu axhunywe ukwenza umugqa ukuze kukhiqizwe ijika elijwayelekile.Ngejika ngalinye elijwayelekile, ukufaneleka kwalo kudinga ukuhlolwa.Inani lemithambeka liwela phakathi kuka--3.3 kanye no-3.8, futhi ukugxila ngakunye kwenziwa ngokuphindwe kathathu.Amaphuzu ahluke kakhulu kwamanye amaphuzu kufanele alahlwe.Inani le-CT lesampula okufanele lihlolwe lilethwa ejikeni elijwayelekile, futhi ileveli yokusho yesampula ezohlolwa ingabalwa.

Inani le-CT lesampula okufanele lihlolwe lilethwa ejikeni elijwayelekile, futhi inombolo yokuqala yekhophi yesampula ezohlolwa ingabalwa.

Ukusebenza kahle kanye neSlope
Umthamo wejika elijwayelekile umele ukusebenza kahle kwe-PCR yesikhathi sangempela.
·I-slope engu--3.322 ibonisa ukuthi ukusebenza kahle kokukhulisa i-PCR kusebenza kahle ngo-1, noma ngo-100%, futhi inani lomkhiqizo we-PCR liphinda kabili emjikelezweni ngamunye.
·Umthambeka ongaphansi kuka--3.322 (isb, –3.8) ukhombisa ukusebenza kahle kwe-PCR
·Umthambeka omkhulu kuno--3.322 (isb, –3.0) ukhombisa ukuthi ukusebenza kahle kwe-PCR kubonakala kukukhulu kuno-100%, okuyisimangaliso, umjikelezo owodwa we-PCR ungenza kanjani okungaphezu kokuphindwe kabili komkhiqizo okhulisiwe?Lesi simo senzeka esigabeni esingesona somugqa sokusabela kwe-PCR, okungukuthi, kunenani elikhulu lokukhulisa okungaqondile.

ijika elincibilikayo
Ngemva kokuqedwa kokukhulisa i-qPCR, umkhiqizo we-PCR uyashisisa.Njengoba izinga lokushisa likhuphuka, umkhiqizo wokukhulisa imigqa emibili uyancibilika kancane kancane, okuholela ekwehleni kokuqina kwe-fluorescence.Uma izinga lokushisa elithile (Tm) lifinyelelwa, inani elikhulu lemikhiqizo lizoncibilika.I-fluorescence yehla kakhulu.Imikhiqizo ehlukene ye-PCR inamanani ahlukene e-Tm namazinga okushisa ahlukene okuncibilika, ukuze kubonakale ukucaciswa kwe-PCR.

Ijika elincibilikayo (ijika eliphuma kokunye)
Ijika elincibilikayo lisuselwa ekwenzeni imephu ephezulu, engabonisa ngokunembile isimo sezingcezu zomkhiqizo we-PCR.Njengoba izinga lokushisa elincibilikayo liyivelu ye-Tm yocezu lwe-DNA, amanye amapharamitha athinta ivelu ye-Tm yocezu lwe-DNA angahlulelwa, njengosayizi wesiqephu, okuqukethwe kwe-GC, njll. Ngokuvamile, ngokwezimiso zethu zomklamo wokuqala,ubude bomkhiqizo okhulisiwe buphakathi kuka-80-300bp, ngakho izinga lokushisa lokuncibilika kufanele libe phakathi kuka-80°C no-90°C.

Ukuhunyushwa kwejika elincibilikayo: Uma ukuphakama okuyinhloko kuphela kuvela phakathi kuka-80°C-90°C, kusho ukuthi i-PCR yomthamo we-fluorescent iphelele;uma isiqongo esiyinhloko sivela phakathi kuka-80°C-90°C neziqongo ezixubile zivela ngaphansi kuka-80°C, i-primer dimer ibhekwa ngokuyisisekelo.Ungazama ukwandisa izinga lokushisa le-anneal ukuze ulixazulule;uma ukuphakama okuyinhloko kubonakala phakathi kuka-80 ° C-90 ° C, futhi ukuphakama okuxubile kubonakala futhi lapho izinga lokushisa likhuphuka, ngokuyisisekelo kubhekwa ukuthi kukhona ukungcola kwe-DNA, futhi i-DNA idinga ukususwa esigabeni sokuqala sokuhlola.

Yebo, kusenezimo ezingajwayelekile, ezizohlukaniswa ngayinye ngayinye ngezansi.
3. Ulwazi oluphambili

Ukwenza i-qPCR, kufanele ngithi MIQE,Ulwazi OluncaneYokushicilelwa kweInaniI-PCR yesikhathi sangempelaUkuhlola—ulwazi oluncane lokushicilela ama-athikili mayelana ne-PCR yobuningi besikhathi sangempelaukuhlola .Ukuze senze ukuqonda kwawo wonke umuntu kube lula, sizokwenza kube lula okuqukethwe okubalulekile.

Ungacinga umbhalo wokuqala we-MIQE ku-inthanethi, futhi okubaluleke kakhulu ukuthi ibekauhlu lokuhlola idatha oludinga ukuhlinzekwa lapho kushicilela i-athikili .

Ababuyekezi bangahlulela ikhwalithi yokuhlolwa ngokufunda le mininingwane;abafundi besikhathi esizayo bangaphinda basebenzise lokhu ukuze baphinde noma bathuthukise isilingo.
Kubalulekile ukuqaphela ukuthi kulolu hlu, ukubaluleka kohlu ngalunye kumakwe ngo-E noma u-D ngokulandelanayo.Kusho ukuthini?E: imininingwane ebalulekile (kufanele ithunyelwe);D: ulwazi olufiselekayo (nikeza okuningi ngangokunokwenzeka).

I-MIQE (1)—Idizayini Yokuhlola
Ama-scumbag amaningi aqede ukuzivikela ngemva kokuphothula izifundo zawo zeziqu ngeke azi ukuthi angaklama kanjani ukuhlola ngokuzimela, avule izincwadi zawo zokubhalela, futhi enze lokho uthisha abatshela ukuthi bakwenze.Ngenxa yalokho, umklamo wokuhlola awuzange uqine, futhi umnyango wokuhlela wephephabhuku wathi bafuna ukwenza lesi sithombe nalesi sithombe, ngakho bakwenza besamangele.Enziwa kanjena ama-scumbags!

Eduze nasekhaya, umgomo wokuqala wokuhlola ukunqumaukuqina komqondo wokuhlola.Into ebaluleke kakhulu idizayini yokuhlola, futhi into ebaluleke kakhulu mayelana nedizayini yokuhlola indlela yokusetha isampuli eqondiwe, isampula yereferensi (ukulawula), kanye nenani lokuphindaphinda, ukuze idatha yokuhlola ikwazi ukukhonjwa, iqhathaniswe, futhi ikholwe.

Isampula okuhlosiweibhekisela kusampula esidinga ukuthi sihlonze isakhi sofuzo esiqondiwe ngemva kokwelashwa okuthile.Isampula yereferensiisampula ngaphandle kwanoma yikuphi ukwelashwa, okuvame ukubizwa ngokuthi uhlobo lwasendle kubhayoloji.

Ukuphindaphinda kokuhlolazibaluleke kakhulu.Ngokuvamile, inani lokuphindaphinda okuhehayo kufanele libe ngaphezu kwezintathu.Kuyadingeka ukuhlukanisa ukuthi kuyini ukuphindaphinda kwebhayoloji nokuthi kuyini ukuphindaphinda kwezobuchwepheshe.

I-Biological Replicates: Ukuhlolwa okufanayo kokuqinisekisa okwenziwe ngezinto ezihlukene (isikhathi, izitshalo, amaqoqo, amapuleti okusabela).

Ukuphindaphinda kwebhayoloji
Ake sithathe ukwelashwa kwezibulala-zinambuzane zikapelepele njengesibonelo.Sifuna ukufafaza izibulala-zinambuzane ezitshalweni ezintathu ze-ABC, izitshalo ezintathu ze-ABC ziyiziphindaphindo ezintathu zebhayoloji, futhi ziwukuhlolwa okufanayo kokuqinisekisa okwenziwa ngezinto ezihlukene.Kodwa njengokuhlola, ukulawula kuyadingeka ngempela, ngakho-ke singafafaza elinye lamagatsha esitshalo A ukuze sakhe iqembu lokuhlola lesitshalo A, futhi singafafazi amanye amagatsha esitshalo A ukuze sakhe iqembu lokulawula.Yenza okufanayo ku-B no-C.

Ukuphindaphinda Kwezobuchwepheshe (Ukuphindaphinda Kobuchwepheshe): Ukuhlolwa okuphindaphindiwe okuklanyelwe ukugwema amaphutha abangelwa ukusebenza, okuyimbobo eyimpinda efakwe kokubalulekile okufanayo.Kokubili ukwelapha nezilawuli kufanele kube nezilungiselelo eziphindaphindayo (ubuncane obuthathu) bofuzo oluqondiwe kanye nofuzo lwangaphakathi lwereferensi.

Ukuphindaphinda kobuchwepheshe
Thatha upelepele ophathwe ngezibulala-zinambuzane njengesibonelo futhi.Eqenjini lokuhlola lesitshalo A, senze izimbobo ezintathu ze-PCR zoku-1, 2, no-3 zofuzo lwazo oluqondiwe kanye nofuzo lwangaphakathi oluyisethenjwa ngokulandelana kwazo, ukuze sithathe isilinganiso ngemva kokutholwa.Ukulawula isitshalo A Amaqembu nawo aphathwa ngendlela efanayo.Ngokufanayo, yenza ukwelashwa okufanayo ezitshalweni zika-B no-C.Lokhu ukuphindaphinda kobuchwepheshe.

Kuyaphawuleka ukuthiokungena ezibalweni ukuphindaphinda kwebhayoloji, futhi ukuphindaphinda kobuchwepheshe ukuhlola ukuthi ingabe zikhona yini izehlakalo ezingahleliwe enqubweni yokuhlola, ukuze kwenziwe imiphumela yokuhlola ithembeke, okungukuthi, ukugwema amaphutha ngokuthatha isilinganiso sawo njengoba sivame ukusho.

Izilawuli Ezingezinhle—NTC kanye ne-NRT
I-NTC (No-Template Control), isilawuli esingenaso isifanekiso, sisetshenziselwa ukuqinisekisa ukuthi into yokuhlola ingcolile yini.Ngokuvamile, amanzi asetshenziswa njengesifanekiso.Uma kukhona ukusabela kwe-fluorescent, kubonisa ukuthi ukungcola kwe-nucleic acid kwenzeke elabhorethri.

Lokhu kungcola kuvela: emanzini angcolile, ama-reagents angafanelekile aqukethe i-endogenous DNA, ukungcoliswa kwe-primer, ukungcoliswa kwemishini yaselabhorethri, ukungcoliswa kwe-aerosol, njll., kudingeka kusetshenziswe ama-scavengers e-RNase kanye nama-RNase inhibitors.Ukungcoliswa kwe-aerosol kunzima kakhulu ukukuthola.Cabanga nje ilabhorethri yakho ifana nentuthu, enama-nucleic acid ahlukahlukene alenga emoyeni.

I-NRT (No-Reverse Transcriptase), isilawuli esingenalo ukuhlehla okulotshiweyo, i-RNA engahlaneki njengokulawula okunegethivu, okuwukulawula kwezinsalela ze-gDNA.

Lapho wenza isisho sofuzo, inani le-RNA litholwa ngokuthola inani le-cDNA ngemva kokuloba okuhlanekezelwe.Uma kunensalela ye-gDNA lapho i-RNA ihlanzwa, izodala amaphutha emiphumeleni yokuhlola, ngoba imiphumela yangempela etholiwe i-gDNA ne-cDNA.Ezingeni elihlanganisiwe, hhayi nje i-cDNA, i-gDNA idinga ukususwa ngokuphelele ngesikhathi sokukhishwa kwe-RNA.

I-MIQE (2)—isampula yolwazi
Ulwazi olubizwa ngesampula lusho ukuthi uma sishicilela indatshana emayelana ne-qPCR, kufanele sichaze imininingwane yesampula ngokucacile, okuyingxenye ebalulekile ye-athikili.Ngokufanayo, lapho sicubungula amasampula, kufanele futhi silawule ukusebenza kwethu ukuze siqinisekise ukufaneleka kwamasampula.

Incazelo yesampula ingumphumela kuphela, futhi kufanele sinake kakhulu izinto ezithathwe phakathi nalo lonke ucwaningo.

Ukukhethwa kwezinto zokuhlola
Amasampula egazi - khetha igazi elisha, hhayi amahora angaphezu kwama-4.Amasampula eseli - khetha ukuqoqa amaseli amasha ngesikhathi sokukhula okunamandla.Izicubu Zezilwane—Khetha izicubu ezintsha, ezikhula ngamandla.Izicubu Zezitshalo - Khetha izicubu ezintsha, ezincane.

Kufanele ukuthi uqaphele ukuthi kunegama elingukhiye kule misho embalwa: fresh .
Kula masampuli angenhla, ikhithi ehamba phambili, engabizi, futhi ezinzile emakethe ikhithi ye-Foregene, engakhipha ngokushesha futhi kalula i-DNA yabo ne-RNA.

Ikhithi encane ye-DNA yegazi

Ikhithi Yokuzihlukanisa Yeseli Ye-RNA

Ikhithi Yokuhlukanisa Yezilwane Ephelele ye-RNA

Ikhithi Yokuhlukanisa Yezitshalo Isamba se-RNA

I-Plant Total RNA Isolation Kit Plus

Ikhithi yokuhlukanisa i-Plant DNA

Ukugcinwa kwezinto zokuhlola
Ngokuvamile, asincomi ukugcina amasampula, uma izimo zivuma.Nokho, kunabangane abaningi abangakwazi ukwenza izivivinyo ngokushesha ngemva kokuthatha amasampula, futhi abanye badinga ngisho nokuthwala amathange e-nitrogen ayiketshezi baye ensimini ukuze kuthathwe amasampula.

Kulolu hlobo lomngane osebenza kanzima, ngingasho nje ukuthi awuqondi izinto ezisetshenziswayo ezisebenzayo.Manje izinkampani eziningi ezisebenzisekayo ezisebenzisekayo zikhiqiza ama-reagents angagcina amasampula e-RNA ekamelweni lokushisa, futhi ungakhetha ukuwasebenzisa.Indlela evamile yokugcina uketshezi lwenitrogen, kusetshenziswa ithangi elincane le-nitrogen eliwuketshezi okulula ukulithwala.Ngemva kokubuyisela isampula elabhorethri, ligcine efrijini elingu -80°C.

Ngokuhlolwa okubandakanya i-RNA, umgomo wamagama ayisithupha kufanele ulandelwe:izinga lokushisa eliphansi, awekho ama-enzyme,futhingokushesha .

Umqondo wokushisa ophansi kulula ukuwuqonda;ngaphandle kwama-enzyme, i-RNase ikuyo yonke indawo emhlabeni esiphila kuwo (kungenjalo ngabe ubulewe i-HIV), ngakho-ke indlela yokugwema i-RNase lapho wenza ucwaningo kuwumqondo obaluleke kakhulu;ngokushesha,Ayikho i-Kung Fu emhlabeni engakwazi ukuphulwa, ijubane kuphela elingenakwephulwa.

Ngakho-ke, ngomqondo othile, lapho isikhathi sokukhipha sisifishane, ikhithi iba ngcono.KunganiI-ForegeneIkhithi igcizelela isivinini, ngoba bayazi kahle.

PS: Amanye amantombazane enza ukuhlola ngokucophelela okukhulu, kodwa awekho kahle njenge-slam dunk ngemva kweminyaka eminingana yomsebenzi.Banomuzwa wokuthi uNkulunkulu akanabulungisa, ukhononda ngabanye futhi ufuna ukuphila.Eqinisweni, wayengakuqondi.Akazange ayivikele kahle i-RNA, kanti nomdlali we-slam dunk ubenenkani.Lapho enza ucwaningo, wayecabanga ukuthi uzoqeda i-slam dunk kathathu, kahlanu kanye nezigaba ezimbili, kodwa wakwenza kahle ukuhlola.

Qaphela: Kancane, amathuba amaningi okuhlasela kwe-RNase.Ungaziqeqesha kanjani ukuze usheshe?Ayikho indlela, vele uzilolonge kakhudlwana.

Ngokuhlolwa okuhlukile namasampuli ahlukene, kusadingeka ukufunda izincwadi eziningi nokukhetha indlela efanelekile yokucubungula.Ngenqubo yokuqoqwa kwesampula nokugcinwa, i-MIQE idinga ukuthi kufanele ibhalwe ngokucacile ephepheni, ukuze ababuyekezi bakwazi ukubuyekeza ukwethembeka kwephepha, futhi kulungele futhi intsha ebambe ongezansi ukuthi iphinde ukuhlola kwakho.

Nakuba ukuhlolwa kwezinto eziphilayo kunzima, kusezingeni eliphezulu.Uma unganakile, ungawugumbuqela umhlaba.Isibonelo, ukwenza i-SARS ibe yinkinga ye-biochemical, noma ukwenza irayisi eyi-hybrid ukusindisa abantu abayizigidi eziyizinkulungwane eziyi-1.3.Isithombe esingezansi siwukuhlolwa kwamakhemikhali, kufanele uqonde ukuthi uziqhenya kangakanani ngocwaningo lwakho ngokubuka nje ukubukeka kwakhe okufana ne-dick.Khohlwa, ungamfaki mnyama.

I-MIQE (3) - isizinda se-nucleic acid.
Ukukhishwa kwe-Nucleic acid kuwumcimbi omkhulu, futhi zonke izivivinyo zebhayoloji yamangqamuzana ziqala ngokukhipha i-nucleic acid.Okokuqala, ake sikopishe okuqukethwe kwe-MIQE ekukhishweni kwe-nucleic acid.

Uma ubheka leli fomu, awukwazi ukuhlala phezulu.Ifomu liyinkolelo-mbono.Ukuze ube umfundi ophambili, kufanele ubuze ukuthi kungani.Okuqukethwe okubalulekile kwaleli thebula yilokhu: Phishekelaubumsulwa, ubuqotho, ukungaguquguquki, kanye nenani lokukhishwa kwe-RNA .

Ingxenye yokuqala yeinqubo noma insimbi isinyathelo sokukhipha i-nucleic acid.Uma usebenzisa i-nucleic acid extractor ukuze ukhiphe (okuthuthukisiwe, ngicela ungithinte ukuze uthenge), udinga ukukhombisa igama lemodeli yethuluzi.

Igama lekhithi kanye

iyiphi ikhithi eyasetshenziselwa imininingwane yoshintsho, iziphi izifudumezi ezikhethekile ezengezwe noma yiziphi izenzo ezikhethekile ezenziwe kufanele zichazwe ngokucacile ukuze abanye baphinde kalula ukuhlola kwakho.

Abanye abantu bengeza ama-reagents athile akhethekile lapho bekhipha amasampula akhethekile, becabanga ukuthi lesi isikhali sabo esiyimfihlo futhi abatsheli abanye.Nakuba beyigcina iyimfihlo, baphinde balahlekelwe ithuba lokwenza isihloko sakho sikhanye.Ungabi ubuhlakani, kufanele uthembeke ngaphezu kwezwe elidala laseZhang ocwaningweni lwesayensi, uma ufuna ubuhlakani, lesi sihloko sizokwenza isilima.

kufanele ukhumbule inombolo yomkhiqizo wekhithiuma u-oda ikhithi bese ubhala isihloko.Ngokuvamile kuba nezinombolo ezimbili kukhithi: Inombolo yekhathalogi yekati (inombolo yomkhiqizo, inombolo ye-athikili), I-Loti—inombolo yenkatho yomkhiqizo ( Isetshenziselwa ukukhombisa ukuthi umkhiqizo uvela kuliphi iqoqo).

Ngaphezu kwalokho, inombolo ye-CAS ivame ukusetshenziswa uma ku-oda ama-reagents amakhemikhali e-biochemical, futhi ngizoyenza idume ndawonye.Inombolo ye-CAS inombolo enikezwe i-American Chemical Society emthini wamakhemikhali omusha ngamunye.Ngokuvamile, izinombolo ezintathu zixhunywe ngodwi.Inombolo ye-CAS kaRushui: 7732-18-5.Amakhemikhali ngokuvamile aneziteketiso eziningi, kodwa inombolo ye-CAS ihlukile.Uma u-oda umuthi, ungabheka inombolo yawo ye-CAS kuqala.

Eduze nasekhaya, kungani kufanele sizichaze lezi zinto ngokucacile?Eqinisweni, futhi ukuhlola ikhwalithi yokukhishwa kwe-RNA.Ukusetshenziswa kwezinsimbi namakhithi kuzokwenza ukukhishwa kwe-RNA kuhambisane.Isilinganiso sokukhishwa kwama-laboratories ajwayelekile asisikhulu, futhi singatholakala ngamakhithi.

Imininingwane yokwelashwa kwe-DNase noma i-RNase
Udaba olubalulekile lwe-fluorescent quantitative PCR ukuvimbela ukungcoliswa kwe-DNA, futhi ungalingi uma kunokungcoliswa.Ngakho-ke, kubalulekile ukusho inqubo oyisebenzise ukucubungula i-DNA, ukuze ubonise ukuthi i-DNA enqubweni yokuhlola isuswe ngokuphelele futhi ngokuphelele.imelelwa umdwebo wohlelo.

Umdwebo we-Schematic we-RNA ne-DNA
Ngokuvamile, indlela yokukhipha i-DNA ukwelapha i-RNA nge-DNase ngemva kokukhipha.Nokho, lezi izindlela ezindala uma kuqhathaniswa.Imishini yokukhipha i-RNA yezentengiso ikwazile ukususa i-DNA phakathi nenqubo yokukhipha ngaphandle kokwengeza i-DNase.Isibonelo, uchungechunge lwamakhithi avela ku-Foregene.

Qaphela: Ukukhipha i-DNA ngesikhathi sokukhishwa kwe-RNA kuyinkemba esika nhlangothi zombili eyingozi kakhulu, ezokwandisa isikhathi sokusebenza sokukhishwa kwe-RNA futhi kwandise ingozi yokuwohloka kwe-RNA.Ngokuyisisekelo, kuwukuhweba phakathi kwesivuno se-RNA nobumsulwa.

Ngaphezu kwalokho, inani le-DNase elingezwe kukholamu ye-adsorption esekelwe ku-silica lincane kakhulu, futhi i-DNase yekhwalithi ephezulu kufanele isetshenziselwe ukuzuza umphumela.I-DNase engalungisiwe ayikwazi ukugaywa ngokushesha nangokuphelele.Lokhu ukuhlola izinga lobuchwepheshe lomthengisi.Yiqiniso, kukhona abathengisi abayinqaba nakakhulu abaziqhayisa ngokuthi i-DNA ingasuswa ngaphandle kwe-DNase.Kungashiwo ukuthi noma ubani ozishaya isifuba ngokuthi i-DNA ingakhishwa ngokuphelele ngaphandle kwe-DNase uyisikhohlakali.I-DNA iyisakhiwo esinemicu ekabili ezinzile, futhi ayinakusulwa ngokukhuluma nokuhleka.

Ukuhlolwa kokungcola
indlela yokuhlola: ukutholwa kwe-electrophoresis, 1% i-agarose, 6V/cm, 15min, ilayisha 1-3 ul

Ukuhlaziywa kobuningi be-Nucleic acid
ngokuvamile ikalwa kusetshenziswa i-spectrophotometer ye-UV.Ake ngiqale ngandise incazelo yamanani amathathu we-OD260, OD280, kanye ne-OD230.
·OD260nm: Iwubude begagasi bokumuncwa kwenani eliphakeme kakhulu lokumuncwa le-nucleic acid, futhi inani elikalwe kangcono lisukela ku-0.1 kuye ku-1.0.Uma kungenjalo, nciphisa noma gxilisa isampula ukuze uyilethe ebangeni.
I-OD280nm: Iwubude beza beza obuphakeme kakhulu bokumuncwa kwamaprotheni nezinto ze-phenolic.
·OD230nm: Iwubude beza beza obuphakeme kakhulu bokumuncwa kwama-carbohydrate.

Okulandelayo, ake sikhulume ngendima yenkomba ngayinye.Ku-A260, ingasetshenziswa ukukala isivuno se-nucleic acid.Lapho OD260=1, dsDNA=50μg/ml, ssDNA=37μg/ml, RNA=40μg/ml.

Ukuze sibe msulwa, sidinga ukubheka izilinganiso esivame ukuzibona: OD260/280 kanye ne-OD260/230.
·I-DNA ehlanzekile: I-OD260/280 icishe ilingane no-1.8.Uma ingaphezu kuka-1.9, ikhombisa ukuthi kunokungcola kwe-RNA, futhi uma ingaphansi kuka-1.6, ibonisa ukuthi kukhona ukungcola kwamaprotheni ne-phenol.
I-RNA ehlanzekile: 1.7
·OD260/230: Kungakhathaliseki ukuthi i-DNA noma i-RNA, inani lesithenjwa ngu-2.5.Uma ingaphansi kuka-2.0, ikhombisa ukuthi kukhona ukungcoliswa kukashukela, usawoti kanye ne-organic matter.

Ubuqotho be-RNA

Kubaluleke kakhulu ukukala ubuqotho be-RNA.Ngokuvamile, kuyadingeka ukwenza isilingo sejeli ye-RNA ukuze uhlole ukuthi ukukhanya phakathi kuka-28S no-18S RNA kuwubudlelwane obukabili.Uma kuvela ibhande lesithathu elithi 5S, kusho ukuthi i-RNA isiqalile ukwehla, ngaphandle kwezilwane ezingenamgogodla.

Idatha yokuhlolwa kwekhwalithi ye-RNA: Ngaphezu kwalokhu kuhlolwa okungenhla, kukhona nokuhlolwa kwezinsimbi ezithuthuke kakhulu ngokuya kobuqotho be-RNA, njengokuhlola ubuqotho be-RQI yohlelo lwe-Experion automatic electrophoresis, olungathola ukuthi i-RNA yonakele ngokungabonakali.

Ocwaningweni lwesayensi, i-fluorescent quantitative PCR iwukuqhathanisa phakathi kofuzo oluqondiwe kanye nofuzo lwangaphakathi oluyisethenjwa.Ngakho-ke, ohlelweni lokulondolozwa kwesampula ye-RNA, ukukhishwa kwe-RNA, njll., umgomo oyinhloko uwukuqinisekisa ubuqotho be-RNA.

Ukuthi ubuqotho be-RNA buthinta kanjani ibhalansi phakathi kofuzo oluqondiwe kanye nofuzo lwangaphakathi lwereferensi kungaqondwa kalula kumfanekiso ongezansi.Ukucekelwa phansi kuzoholela ekungapheleleni kofuzo, kungakhathaliseki ukuthi ukungapheleli kofuzo lwangaphakathi lwereferensi noma ukungapheleli kofuzo oluqondiwe, kuzoba nomthelela omkhulu kudatha.

Umdwebo wohlelo lofuzo oluqondiwe kanye nofuzo lwereferensi, akumele kube yiqiniso

Ukuhlolwa kokuvimbela (ukuthi inani le-CT licindezelwe ngaphansi kokugxilisa okuphakeme noma okuphansi noma ezinye izimo)

Uma sithatha lesi sibalo njengesibonelo, amanani we-Ct wamajika amahlanu alandelayo.Ukusatshalaliswa kwamanani e-CT phakathi kwamajika akulingani, futhi amanani e-Ct abambezeleka ngaphansi kokugxila okuphezulu nokuphansi, okuyindaba yokuvinjelwa kwe-PCR.

Iphuzu elibalulekile: Enqubweni yokukhipha i-RNA, sidinga ukushiya imibono eyiphutha bese sisungula elungile.

Umbono ongalungile uthi: Ukukhishwa kwe-RNA kulandela isivuno kuphela, becabanga ukuthi uma likhulu inani le-RNA elitholiwe, lingcono.Eqinisweni, uma senza ukulinganisa, uma inani lezakhi zofuzo lingelikhulu kakhulu, asidingi i-RNA eningi.Inani le-RNA oyikhiphayo lingaphezu kokwanele.

Umqondo olungile uthi:Ukukhishwa kwe-RNA kufanele kuphishekele ukuhlanzeka, ubuqotho nokungaguquguquki.Ukuhlanzeka kungaqinisekisa ukuthi okulotshiweyo okuhlanekezelwe okulandelayo akuvinjwa futhi idatha ngeke ithintwe i-DNA.Ubuqotho buqinisekisa ibhalansi yokulandelana okuqondiwe kanye nezinkomba zangaphakathi.Ukuvumelana kuqinisekisa ukulayishwa kwesampula okuzinzile.

I-MIQE (4) – ukuloba okuhlanekezelwe
Umbono oyiphutha: ukuphishekela umthamo wesampula ophezulu.
Umqondo olungile: Phishekela ukuvumelana (ukuzinza), kungakhathaliseki inani le-RNA elilayishiwe, ukusebenza kahle kokulotshwa okuhlanekezelwe kuhlala kufana, kuqinisekisa ukuthi umehluko ku-cDNA ungabonisa ngempela umehluko ku-mRNA.
Sichaza le nqubo ngomdwebo wohlelo:

Umdwebo wohlelo lokusebenza kokuhlehla kokuloba, ungabi yiqiniso
Okokuqala, sidinga ukuqonda umehluko phakathi kwenqubo yokuhlehla yokulotshwa kanye nenqubo ye-PCR.I-PCR ingena ezinqubweni eziningi zokushisisa nokufaka amanzi, futhi ingxenye eqondiwe ikhula ngamandla;ngenkathi ukuloba okuhlanekezelwe kungenayo le nqubo, singacabanga ukuthi ukuloba okuhlanekezela empeleni kungukukodwa kokukodwa Phakathi nenqubo yokuphindaphinda, izingcezu eziningi ze-RNA.

njengoba kukhona bangathola izingcezu eziningi zolwazi lwe-cDNA, kufanele kuqondwe manje, ngoba izingcezu ezinkulu nezincane zilotshwe ngokuhlehla, futhi akunakwenzeka ukugxila kucezu olulodwa.Futhi ngenxa yokuthi inani le-RNA lincane uma kuqhathaniswa, inani le-cDNA elitholiwe nalo lincane ngokuqhathaniswa, ngokungafani ne-PCR, enomphumela wokukhulisa, ngakho-ke akwenzeki ukubonwa.

Imiphumela ye-cDNA electrophoresis
Okwesibili, kuhle, ukulotshwa okuhlanekezelwe kwenziwa umuntu nomuntu, kodwa akukho okulotshiweyo okuhlanekezela okuvela kunoma iyiphi inkampani okungafinyelela lo mphumela.Ngokuyisisekelo, ukusebenza kahle kwama-transcriptases amaningi ahlanekezela kuzulazula phakathi kuka-30-50%.Uma kunjalo, singathanda ukuba nokusebenza kahle kokuhlehla okuzinzile uma kuqhathaniswa, okuyikhona esifuna ukukubona emfanekisweni: Ama-RNA ama-3 athola ama-cDNA angu-2, ama-RNA angu-6 athola ama-cDNA angu-4, ngakho-ke kungakhathaliseki ukuthi isampula elingakanani lilayishiwe, ukusebenza kahle kokuhlehla kokuloba kuzinzile uma kuqhathaniswa.Asifuni ukubona isimo lapho ukusebenza kahle kokuloba okuhlanekezelwe kungazinzi futhi ukugxila okuphezulu kuvinjiwe.

Ngakho-ke, ungaqinisekisa kanjani ukuthi ukusebenza kahle kokuhlehla kokuloba kuzinzile?Indlela ilula kakhulu, udinga kuphela ukwenza ukuhlolwa kokuqhathanisa: enye iwukuba uhlehlise ukuloba ku-cDNA ngemva kokuhlanjululwa okuphindwe kabili kwe-RNA, kanti enye iwukwenza ukuhlanjululwa okuphindwe kabili ngemva kokuloba okuphambene ku-cDNA, bese wenza i-qPCR ukuze ubone ukuthambeka okutholiwe Ingabe kuyavumelana.Njengomfundi ophakeme, kufanele ukuqonde ngemizuzwana.Njengoba kuboniswe ngezansi:

Ukuhlanjululwa kwe-RNA ne-cDNA ukuhlola ukuthi ukusebenza kahle kokulotshwa okuhlanekezelwe kuzinzile yini
Reverse transcriptase kanye nekhithi
I-PCR ye-fluorescent quantitative ephelele ingaba kanjani ne-reverse transcriptase nekhithi enhle kakhulu.I-Reverse transcriptase ihlukaniswe yaba izinhlobo ezimbili ngokuya ngomthombo, i-AMV nomaI-M-MLV, futhi ukusebenza kwazo kuyafana nalokho okuboniswe etafuleni.

Umsebenzi we-RNase H
I-RNase H iyi-Ribonuclease H, igama lesiShayina i-ribonuclease H, okuyi-endoribonuclease ekwazi ukwenza i-RNA i-hydrolyze ngokukhethekile ochungechungeni lwe-DNA-RNA hybrid.I-RNase H ayikwazi ukwenza i-hydrolyze amabhondi e-phosphodiester ku-DNA enomucu owodwa noma ephindwe kabili noma i-RNA, okungukuthi, ayikwazi ukugaya i-DNA enomucu owodwa noma ephindwe kabili noma i-RNA.Okuvame ukusetshenziswa ekuhlanganiseni umucu wesibili we-cDNA.

Kuyinto engavamile.Sithi i-reverse transcriptase inomsebenzi we-RNase H, hhayi ukuthi i-reverse transcriptase iqukethe i-RNase H, futhi kungase kungenzeki ukuhlukanisa i-RNase H ne-reverse transcriptase, mhlawumbe ngenxa yokuhlangana kwamaqembu athile ku-reverse transcriptase Lo msebenzi ubangelwa i-reverse transcriptase.

Ngakho-ke, kungakhathaliseki ukusebenza kahle okuphezulu kokuhlehla kokuloba kwe-AMV, umsebenzi wayo we-RNase H wehlisa isivuno se-cDNA.Kunjalo, abakhiqizi bezinto ezisizayo bahlala bethuthukisa imikhiqizo yabo ukuze baqede umsebenzi we-RNase H ku-reverse transcriptase ngangokunokwenzeka ukuze bakhulise isivuno se-cDNA.
Izinga lokushisa le-anealing

Isakhiwo sesibili se-RNA emazingeni okushisa ahlukene
Bona isibalo esingenhla sesakhiwo sesibili se-RNA emazingeni okushisa ahlukene, futhi usebenzise ithuluzi le-inthanethi le-mFold ukuze unqume ukwakheka kwesibili kwesiqephu esiqondiwe ngaphansi kwezimo ezithile zokushisa nezimo zokugxilisa usawoti.Ku-55 ° C, isakhiwo sesibili se-RNA sisayinkimbinkimbi kakhulu, i-reverse transcriptase ayikwazi ukusebenza, futhi isakhiwo sesibili asikwazi ukuxazululwa ngokuphelele kuze kube ngu-65 ° C, kuyilapho izinga lokushisa eliphezulu le-AMV ne-M-MLV liphansi kakhulu kunalokhu kushisa.
okufanele ngikwenze?Isakhiwo sesibili ukumataniswa okuhambisanayo kwesifanekiso ngokwaso, okuholela ekuqhudelaneni okuqinile phakathi kwe-primer ne-reverse transcriptase nesifanekiso, okuholela ochungechungeni lwezinkinga ezifana no-E ophansi nokuphindaphinda okubi.

okufanele ngikwenze?Khulisa kuphela izinga lokushisa le-annealing ngangokunokwenzeka .

Abakhiqizi abaningi be-reagent bathuthukisa i-reverse transcriptase ngokusebenzisa ubunjiniyela bofuzo.Abanye bakhuphula izinga lokushisa lokusabela, njenge-Jifan ne-Aidelai, futhi abanye basusa iqembu elisebenzayo le-enzyme ye-RNase H ukuze kuthuthukiswe ukuhlobana phakathi kwe-enzyme nesifanekiso se-RNA.Ukusondelana okuphezulu kungacindezela ngokuncintisana kuphume ukwakheka kwesibili futhi kufundwe kahle, futhi kuthuthukise kakhulu ukusebenza kahle kokuhlehla kokuloba.
Iphuzu elingukhiye: Ukuhlehla okulotshiweyo kubaluleke kakhulu ukuphishekela ukuvumelana kokuhlehla kokubhala kahle (ama-enzyme akumele asebenze kahle kuphela kodwa futhi azinze), kunenani lesampula elilayishiwe, uma kungeyona i-PCR yomthamo omkhulu we-fluorescent, ngeke kwenzeke nhlobo.Ama-cDNA amaningi.
Abakhiqizi abahlukahlukene nabo benze imizamo ethile ekuphishekeleni ukungaguquguquki.Isibonelo, izinkampani eziningi manje sezipakishe ukuloba okuphambene njengekhithi evamile ethengiswayo, okuyisinqumo esihle.
Isibonelo, amakhithi kaForegene's RT Easy Series:

I-RT Easy I(Master premix yekhithi yokuqala ye-cDNA synthesis kit)

I-MIQE (5) - ulwazi lofuzo oluqondiwe

Umfanekiso ongenhla uyachaza
1. Ukuthi lolu fuzo luyasebenza yini ekuhloleni okuphindaphindiwe ngokuvamile lungaqinisekiswa ngokuhlolwa okuphindaphindiwe.
2. I-Gene ID, uyazi.
3. Ubude bofuzo, ubude bengqikithi yofuzo oluqondiwe abunankinga nakanjani.Lapho uklama ama-primers, qinisekisa ukuthi ubude be-amplicon buphakathi kuka-80-200bp ukuze uqinisekise ukusebenza kahle kokukhulisa ukuphakama.
4. Ulwazi lokuqhathanisa lokuqhuma okulandelanayo, isakhi sofuzo esiqondiwe sidinga ukuqhathaniswa ku-genebank ukuze kuvinjelwe ukukhuliswa okungaqondile.
5. Ukuba khona kwama-pseudogenes.I-pseudogene iwukulandelana kwe-DNA efana nesakhi sofuzo esivamile kodwa ilahlekelwa umsebenzi wayo ovamile.Ivame ukuba khona emndenini wezakhi eziningi ze-eukaryote.Ngokuvamile imelelwa ngu-ψ.Ikhophi ye-DNA ye-genomic engasebenzi ku-genome efana kakhulu nokulandelana kofuzo lwekhodi., ngokuvamile azilotshiwe, futhi azinayo incazelo ecacile yokuphila komzimba.
6. Ukuma kwama-primers ahlobene nama-exons nama-intron.Eminyakeni yokuqala, lapho sixazulula inkinga yokungcoliswa kwe-DNA, sasivamise ukunaka izindawo zama-primer, ama-exons, nama-intron, futhi ngokuvamile sicabangele ukuklama ama-primer kuwo wonke ama-intron ukuze sigweme ukukhuliswa kwe-DNA.Sicela ubone umfanekiso ongezansi: omnyama umele ama-intron, aluhlaza okwesibhakabhaka ahlukahlukene amele ama-exons, ipinki imele ama-primer ajwayelekile, futhi okubomvu okukhanyayo kumelela ama-intron-spanning primer.

I-Schematic, akulona iqiniso
Yeka ukuthi lokhu kubonakala kuwuhlelo oluphelele kangakanani, kodwa empeleni, ezimweni eziningi, ama-trans-intron primers awawona umlingo njengoba kucatshangwa, futhi azophinde abangele ukukhuliswa okungaqondile.Ngakho indlela engcono kakhulu yokuvimbela ukungcoliswa kwe-DNA ukukhipha i-DNA ngokuphelele.
7. Isibikezelo sokuvumelana.Usebenzisa lesi sibonelo futhi, sebenzisa ithuluzi le-inthanethi le-mFold ukuze unqume ukwakheka kwesibili kocezu oluqondiwe ezingeni elithile lokushisa kanye nokugxiliswa kukasawoti.

Isakhiwo sesibili se-RNA emazingeni okushisa ahlukene
Isakhiwo sesibili ukubhanqa okuhambisanayo kwesifanekiso ngokwaso, okuzoholela ekuqhudelaneni okuqinile phakathi kokumataniswa kwe-primer nesifanekiso, futhi amathuba okubopha i-primer mancane, okuholela ochungechungeni lwezinkinga ezifana no-E ophansi kanye nokuphindaphinda okubi.Ngokubikezela kwesoftware, uma ingekho inkinga yesakhiwo sesibili, lokho kungaba kuhle.Uma kukhona, isihloko sethu sokulandelela sizoxoxa ngokuqondile ngokuthi le nkinga ingaxazululwa kanjani.

I-MIQE (6)—qPCR Oligonucleotides

Ku-PCR ye-fluorescent quantitative, into yokuqala ozabalaza nayo nsuku zonke ukukhipha kwe-RNA, futhi into yesibili kungase kube umklamo wokuqala.
Okokuqala nje, sisahlola imithetho mayelana ne-primer design ngokohlu lokuhlola lwe-MIQE.Kulula kangangokuthi ama-scumbags angakwazi ukuhleka, futhi singakuqeda ngomusho owodwa: thola ukulandelana nokuma kwe-primer probe kanye nendlela yokuguqula.Ngendlela yokuhlanza i-primer, ukuhlanganiswa kwe-primer ishibhile kakhulu njengamanje, i-qPCR ifanele i-PAGE nangaphezulu nezindlela zokuhlanza, futhi ulwazi lwethuluzi lokuhlanganiswa alubalulekile.Abantu abaningi bebelokhu benza ama-primer amashumi eminyaka futhi abazi ukuthi i-synthesizer yi-ABI3900.
Mayelana nezimiso zokuklama kokuqala, akudingekile ukuba uzibambe ngekhanda ngekhanda, ngoba isofthiwe eminingi yokuklama i-primer noma amathuluzi aku-inthanethi angakwazi ukunakekela lezi zinkinga (okunconyiwe kwethuluzi le-inthanethi primer3.ut.ee/), futhi u-99.999% womklamo wokuqala awenziwa ngesandla Bheka, umbhali ngezinye izikhathi uklama amakhulukhulu ezinto zokuqala ngosuku, uma ufunda ngayinye ngayinye, izophambana nayo.
Vele uhlole amaphuzu alandelayo ngemva kokuklanywa kokuqala:
1. Iziqalo zedizayini eziseduze nokuphela kwe-3′: Esimeni sokusebenzisa ama-primers we-oligo dT we-cDNA yokuqala-strand synthesis, kucatshangelwa ukusebenza kahle kokubhala okuhlanekezela kanye nobuqotho be-RNA, ama-primer aklanyelwe adinga ukuklanywa eduze nesiphetho se-3′ ukuze kuthuthukiswe ukusebenza kahle kokukhulisa ukukhulisa.Sebenzisa isithombe ukuchaza kanje (ayikho indlela yokuqonda lokhu):

Kungani ama-primers kufanele aklanywe eduze nokuphela kwe-3′, akumele kube yiqiniso
2. Inani le-TM: Inani le-Tm liku-55-65°C (ngoba umsebenzi we-exonuclease uphakeme kakhulu ku-60°C), futhi okuqukethwe kwe-GC kuku-40% -60%.
3. UKUQHUBEKA: Ukuze ugweme ukukhuliswa okungaqondile kwe-genome, i-Blast kufanele isetshenziselwe ukuqinisekiswa okungeziwe.

I-MIQE(7)—qPCR inqubo

1. Ikhithi ye-qPCR
Ngokwezidingo ze-MIQE, kufanele sichaze ngokucacile izimo zokusabela eziphelele esihlokweni, okuhlanganisa ukucushwa kwesistimu yokusabela ye-PCR, iyiphi ikhithi esetshenziswayo, ubani umenzi, likhulu kangakanani uhlelo lokusabela, kungakhathaliseki ukuthi kusetshenziswa indlela yokudayi noma indlela yophenyo, izilungiselelo zohlelo lwe-PCR.Abashayeli abamakadebona bazothola ukuthi inqobo nje uma ikhithi ikhethiwe, ulwazi olungenhla lunqunywa ngokuyisisekelo.
Njengamanje, ukukhiqizwa nokukhiqizwa kwamakhithi e-PCR e-fluorescent kuwubuchwepheshe obuvuthwe kakhulu.Uma nje ungakhethi abakhiqizi ababi kakhulu, amathuba ezinkinga awaphezulu, kodwa sisafuna ukwabelana nawe ngamaphuzu ambalwa:
I-Taq enzyme yokuqala eshisayo:Ingxenye ebaluleke kakhulu ye-PCR yi-enzyme ye-Taq eqala ukushisa.Ama-enzyme aqala ukushisa emakethe ngokuvamile ahlukaniswe abe izinhlobo ezimbili, eyodwa iyi-enzayimu eshintshwa ngamakhemikhali eqala ukushisa (ungacabanga ukuthi ishumeka upharafini), kanti enye ithiUkuguqulwa kwamakhemikhali kuyindlela yokuqala yama-enzyme aqala ukushisa.Lapho izinga lokushisa elithile lifinyelelwa, i-enzyme izokhulula umsebenzi wayo.I-antibody-modified hot-start enzyme isebenzisa izindlela zebhayoloji ukuvimba umsebenzi we-enzyme.Lapho izinga lokushisa elithile lifinyelelwa, i-antibody izokhishwa futhi ingasebenzi njengeprotheni, futhi umsebenzi we-enzyme uzokwenziwa.

Nokho, yini ukusetshenziswa kwalokhu?Kulokhu, umsebenzi wokukhishwa kwama-enzyme aguqulwe amasosha omzimba uyashesha kunalowo wama-enzyme aguqulwe ngamakhemikhali, ngakho-ke ngokuya ngokuzwela, ama-enzyme aguqulwe ngama-antibody anenzuzo encane, ukuze awekho ngokuyisisekelo ama-enzyme aguqulwe amakhemikhali ezikhithini emakethe.Uma kukhona, khona-ke ubuchwepheshe balo mkhiqizi busabambelele enkathini yenkulungwane yeminyaka.
Ukuhlushwa kwe-Magnesium ion:Ukuhlushwa kwe-Magnesium ion kubaluleke kakhulu ekuphenduleni kwe-PCR.Ukugxila kwe-magnesium ion efanele kungakhuthaza ukukhululwa komsebenzi we-Taq enzyme.Uma ukugxila kuphansi kakhulu, umsebenzi we-enzyme uzoncishiswa kakhulu;uma ukugxila kuphezulu kakhulu, i-enzyme-catalyzed non-specific amplification izothuthukiswa.Ukuqoqwa kwama-ion e-magnesium kuzophinde kuthinte ukufakwa kweziqalo, izinga lokushisa elincibilikayo lesifanekiso kanye nemikhiqizo ye-PCR, ngaleyo ndlela kuthinte isivuno sezingcezu ezikhulisiwe.Ukuhlushwa kwe-magnesium ion ngokuvamile kulawulwa ku-25mM.Yiqiniso, ukuze kube nekhithi enhle, ukugxila kwe-magnesium ion kufanele kulawulwe kahle.Abanye abathengisi bengeza i-ejenti ye-magnesium ion chelating ku-reagent, engafinyelela umphumela wokulungiswa okuzenzakalelayo kokugxiliswa kwe-magnesium ion.
Ukugxiliswa kodayi we-Fluorescent:Udayi we-fluorescent, okuwu-SYBR Green esivame ukuwusebenzisa, ikakhulukazi ukhiqiza i-fluorescence ngokubophezela ku-groove encane ye-DNA enemicu ephindwe kabili, ngoba ukubopha kukadayi ku-DNA enemicu ephindwe kabili akuqondile, okusho ukuthi Inqobo nje uma i-DNA enemicu ephindwe kabili ihlanganiswa nayo, i-DNA ingakwazi ukuhlanganisa i-fluore ne-fluore yakha isignali yangemuva.
PS: Ngenxa yezindawo zayo ezingezwani nokukhanya, imikhiqizo emakethe ivamise ukupakishwa ngamashubhu ensundu opaque centrifuge (njengoba kukhonjisiwe esithombeni esingezansi).Nokho, lokhu kuzohlangabezana nenkinga.Kunzima ukubona ukuthi uketshezi luyamuncwa yini uma kwenziwa isampula.Kulokhu, i-Qingke iyona esebenziseka kalula kakhulu (njengoba kukhonjisiwe esithombeni esingezansi), futhi ishubhu ebonisa ngale ihlanganiswe esikhwameni sikathayela opaque.Bese uyifaka esikhwameni sikathini, ucabangela ukuthi kulula ukugwema ukukhanya nokuthatha amasampula.Kufanele ukhethe inombolo yomkhiqizo efanele.I-TSE204 ukuphila okungabizi kakhulu, okungenza ngifune ukutshala utshani.

Ukugxila kodayi we-fluorescent nakho kubaluleke kakhulu.Uma ukugxila kuphansi kakhulu, ijika lokukhulisa ngeke likhuphuke esigabeni sakamuva futhi aliphelele;uma ukugxila kuphezulu kakhulu, kuzodala ukuphazamiseka komsindo.Njengoba i-PCR yobuningi be-fluorescent incike kakhulu enanini le-CT, uma ukugxiliswa kukadayi we-fluorescent kungalungiswa kahle, indawo ephansi ingcono kunephoyinti eliphezulu.Yiqiniso, ukugxilwa kodayi okufanelekile kungcono kakhulu.

I-ROX: Odayi be-ROX basetshenziselwa ukulungisa amaphutha esignali ye-fluorescence kahle ukuya kahle.Abanye abakhiqizi bezinsimbi badinga ukulinganisa, kanti abanye abakwenzi.Isibonelo, ukusetshenziswa kwethuluzi lokukhulisa i-Thermo Fisher Scientific's Real Time PCR ngokuvamile kudinga ukulinganisa, okuhlanganisa 7300, 7500, 7500Fast, StepOnePlus, njll. Imiyalo yekhithi evamile izoyichaza.
I-Foregene's qPCR Mix iqukethe nodayi we-ROX, olungele ukusetshenziswa kumamodeli ahlukahlukene.

Isikhathi Sangempela PCR Kit-Taqman

Ukwelashwa kwebhondi ye-hydrogen ebuthakathaka: Ukwelashwa kwamabhondi e-hydrogen abuthakathaka kuyindaba yobuchwepheshe uma kuqhathaniswa.Akukho okufunde izincwadi zamakhithi amaningi, kodwa akukho neyodwa yazo ekhulume ngalesi sihloko.Eqinisweni, kubaluleke kakhulu.Ukuhlanganiswa kwezisekelo ikakhulukazi kuncike emandleni e-hydrogen bond.Amabhondi e-hydrogen aqinile awukukhulisa okuvamile, futhi amabhondi e-hydrogen abuthakathaka aholela ekukhuliseni okungaqondile.Uma amabhondi e-hydrogen abuthakathaka engenakuqedwa kahle, ukukhuliswa okungaqondile akukwazi ukugwenywa.Ngaphakathi kobubanzi bombhali, izinkampani ezimbalwa kuphela eziyibonile le nkinga.Uma uthenga ikhithi, ungabheka ukuthi ngabe uke wacabanga yini isisombululo kule ndaba yekhithi ofuna ukuyikhetha.

Ivolumu yokusabela: Uhlelo lwe-20-50ul lusetshenziswa kakhulu, futhi amavolumu amancane angase abangele amaphutha.Ngokuvamile, imiyalelo yekhithi izoncoma ukusetshenziswa kwevolumu yokusabela kwe-PCR.Ungahlakaniphile futhi usebenzise amavolumu amancane ukuze wonge izindleko.umgomo we.Ivolumu enconywe abathengisi ihlolwe ngempela, futhi kungenzeka ukuthi abakwazi ukuxazulula inkinga yamaphutha abangelwa amavolumu amancane.
2. Umkhiqizi kanye nenombolo ye-athikili yepuleti leshubhu
Wonke umuntu uyazi isimiso se-fluorescent quantitative PCR.Ukuqoqwa kwe-fluorescence kwenziwa ikakhulukazi ngama-PCR tube caps.Lapho ukhetha izinto ezisetshenziswayo ze-PCR, naka amaphuzu amabili: ukudluliswa kokukhanya okuhle futhi kulungele ithuluzi.Ngokuvamile, amabhodi namashubhu emikhiqizo evamile alungile, kodwa kufanele ukhethe ngokucophelela mayelana nokujwayela, ngaphandle kwalokho ngeke ukwazi ukusebenzisa ithuluzi.

4. Ulwazi lwezinga eliphezulu

I-MIQE (8)—qPCR ukuqinisekiswa
Lokhu okuhamba phambili okuphezulu kwe-qPCR!Maningi-ke amaqhawe awele esihlabathini lapha.Yebo, kungenzeka futhi ukuthi unenhlanhla futhi izakhi zofuzo ozifundile zilula, ngakho wantanta emhumeni weqhwa eduze nomoya.Ulwazi lokuqinisekisa lwe-qPCR luhloselwe ukuhlola ukwethembeka kwedatha.Sibhala imininingwane yokuqinisekisa edingekayo ngale ndlela elandelayo:

1.Ukuhlolwa kokucaciswa
Ukucaciswa kokukhulisa isakhi sofuzo okuhlosiwe kuhlolwa ngokubheka ukuthi isithombe se-electrophoresis siyibhande elilodwa yini;ukulandelana kokuqinisekisa;ijika elincibilikayo ukuze ubone ukuthi imephu ephezulu ayiyona yini eyodwa;ukuqinisekiswa kokugaya kwe-enzyme nezinye izindlela.
Lapha, sigxila ku-tuhlaziya ukukhulisa okungaqondile kusetshenziswa indlela yokuncibilikisa amajika.Ngokuvamile, lapho siklama ama-primers, usayizi wocezu lomkhiqizo udingeka ukuthi ube sebangeni elingu-80-200bp, okwenza izinga lokushisa elincibilikayo lomkhiqizo we-PCR libe ngu-80-85 °C ububanzi.Ngakho-ke, uma kukhona iziqongo ezixubile, kufanele kube neminye imikhiqizo yokukhulisa engaqondile;uma inani eliphakeme livela ngaphansi kwama-80 ° C, ngokuvamile libhekwa njenge-primer dimer;uma isiqongo sivela ngaphezu kuka-85°C, ngokuvamile sibhekwa njengokungcoliswa kwe-DNA noma ukukhulisa okungaqondile okungaqondile kwezingcezu ezinkulu.
Qaphela: Ngezinye izikhathi kuba khona ukuphakama okukodwa kuphela ku-80°C.Ngalesi sikhathi, lo mqondo kufanele ulandelwe.Kungenzeka ukuthi imiphumela yokukhulisa wonke ama-primer dimers.

Ijika elincibilikayo elivamile (isiqongo esisodwa esingenaso isandiso esingaqondile)

Ijika elincibilikayo elinenkinga (ukukhulisa okungaqondile kweziqongo ezingamanga)
【Ukuhlaziywa kwecala】

Kukhona isiqongo esikhulu, kodwa i-primer dimer ibucayi
Ijika elincibilikayo elinesiqongo esisodwa emfanekisweni ongezansi lingakhohlisa kalula amehlo akho, licabange ukuthi ukuhlola okuphelele, kodwa umphumela awulungile neze.Ngalesi sikhathi, kufanele sibheke izinga lokushisa elincibilikayo.Izinga lokushisa eliphakeme lingaphansi kuka-80°C, okuyi-primer-dimer ngokuphelele.

Alukho ucezu oluqondiwe, wonke ama-primer dimers
Lapha, umfowethu akakwazi ukuma.Lesi sithombe esingezansi yisithombe esithathwe nomakhalekhukhwini engithunyelelwe wona udoti.Ama-reagents awasebenzisile wonke amabhrendi asetshenziswa kakhulu embonini.Washintsha kusuka kolunye uhlobo lwesiqalo se-T waya kolunye uhlobo lwesiqalo se-T.Ngicabanga ukuthi usuvele ukuqagelile.Isikhohlakali sangikhalela: “Isisetshenziswa esisetshenziswe esithombeni sokuqala sihle kakhulu, futhi isiqongo sisodwa.Kamuva, ngemva kokusebenzisa i-reagent oyincomile, iba njengesithombe sesibili, esineziqongo ezixubile.Ungiphathe kabi.“
Hlukanisa amagrafu amabili.Uma uthi nhlá, enye inesiqongo esisodwa, kanti enye inesiqongo esiphindwe kabili.Umbhedo, ukuphakama okukodwa kulungile.Ingabe kuyiqiniso lokho?
Okubi kakhulu kunoDou E, uma ngibeka izithombe ezimbili esithombeni esingezansi, uzoqonda ngokushesha.Eqinisweni, sikhubazeka kalula yilolu hlobo lwesithombe.Ngemva kokuhlaziya ngokucophelela, sithole ukuthi: ukuphakama kwesibalo sokuqala ku-75 ° C, okuyi-primer dimer ngokuphelele;inani eliphakeme lesibalo sesibili livela ku-75 ° C no-82 ° C, okungenani kukhona Umkhiqizo uvela.

Izithombe zempendulo zabafundi
Ngakho-ke inkinga eyisisekelo akuyona inkinga yama-reagents, kodwa inkinga ye-primer design.Ngesikhathi esifanayo, kufakazela futhi ukuthi ezinye izinhlobo ezinkulu aziyona ikhwalithi yensimbi, futhi kufakazela lokho umfowethu akusho ngaphambili: Akulona uhlobo lwe-reagent olusekela isihloko sakho.Yisihloko sakho esisekele uhlobo lwama-reagents.Cabanga nje, uma i-scumbag ingashintshi ama-reagents, idatha engalungile izothunyelwa kumagazini, futhi okuzokwenzeka kungaba yinhlekelele.
2. Inani le-Ct lokulawula okungenalutho
Ungachazi, uma isilawuli esingenalutho sinenani le-Ct, akukhona yini ukungcola?Nokho, kusadingeka uqonde ukuthi yikuphi ukulawula okungenalutho okunenani le-Ct.Uma kuyi-NTC, kusho ukuthi kune-DNA yangaphandle njengokungcoliswa kwe-reagent.Uma kuyi-NRT, kusho ukuthi i-RNA ekhishiwe inokungcola kwe-DNA.
3. Ijika elijwayelekile
Kubandakanya umthamo kanye nefomula yokubala, ukusebenza kahle kwe-PCR kungabalwa ngefomula.Ukuhlolwa okuphelele kudinga ukuthambeka kwejika elijwayelekile ukuze usondele ku-3.32, kanye no-R² ukuze usondele ku-0.9999.
4. Ububanzi obuguquguqukayo bomugqa
Ububanzi obuguqukayo bokusabela buwumugqa.Ngokwethempulethi esetshenziselwa ukukhiqiza ijika elijwayelekile, ububanzi obuguqukayo kufanele bufake okungenani ama-concentration gradients angu-5, futhi sinake ukuguqulwa kwamanani e-Ct kuma-gradients aphakeme kanye namagradient okugxilisa ingqondo aphansi.
5. Ukunemba kokutholwa
Izinguquko emiphumeleni ye-qPCR, okungukuthi, ukuphindaphinda okubi, okungukuthi, ukunganembi kahle, kubangelwa izici eziningi, ezihlanganisa izinga lokushisa, ukugxilisa ingqondo, nokusebenza.Ukunemba kwe-qPCR kuvame ukulawuleka kancane njengoba inombolo yekhophi iyancipha.Ngokufanelekile ngaphakathi-kokuhlukahluka kokuhlola, lokhu kuhluka kobuchwepheshe kufanele kuhluke ekuhlukeni kwezinto eziphilayo, futhi okuphindaphindwayo kwebhayoloji kungabhekana ngqo nomehluko wezibalo emiphumeleni ye-qPCR phakathi kwamaqembu noma ukwelashwa.Ikakhulukazi ekuhlolweni kokuxilonga, ukunemba okungcono kakhulu kwe-inter-assay (ukuphindaphinda) kuwo wonke amasayithi kanye nabasebenzisi kufanele kubikwe.
6. Ukusebenza kahle kokutholwa kanye ne-LOD (ku-multiplex qPCR)
I-LOD iwukugxila okuphansi kakhulu kwamasampuli angama-95% atholiwe.Ngamanye amazwi, ukugxila kwe-LOD equkethwe phakathi kwesethi yeziphindaphinda zofuzo eziqondiwe akufanele kudlule u-5% wokusabela okuhlulekile.Lapho kwenziwa ukuhlaziya kwe-multiplex qPCR, ikakhulukazi ukuze kutholwe ngesikhathi esisodwa ukuguqulwa kwamaphoyinti noma ama-polymorphisms, i-multiplex qPCR idinga ukunikeza ubufakazi bokuthi ukunemba kwezingcezu eziningi eziqondiwe akonakali kushubhu efanayo, ukutholwa okuningi kanye nokutholwa kweshubhu elilodwa Ukusebenza kahle kanye ne-LOD kufanele kufane.Ikakhulukazi lapho izakhi zofuzo eziqondiswe ekugxilweni okuphezulu kanye nezakhi zofuzo eziqondiswe ekugxilweni okuphansi zikhuliswa kanyekanye, le nkinga kufanele inakwe.
Izinkinga nezisombululoNgokuvamile, izinkinga ezivame ukuhlangana nazo ekulungiseni iphutha kwe-qPCR zigxila kulezi zici ezilandelayo:
·ukukhulisa okungaqondile
·Ukukhetha okunzima kokugxila kwe-primer kanye nenkinga ngama-primer-dimers
·Izinga lokushisa le-anneal alilungile
·Isakhiwo sesibili sithinta ukusebenza kahle kwe-amplification
ukukhulisa okungaqondile
ukukhulisa okungaqondilekwenzeka , ngokuvamile kubhekwa ukuthi umklamo we-primer awufanelekile yini, kodwa uma ungajahi ukushintsha iziqalo, ungazama izindlela ezilandelayo kuqala (isimiso siphinde sinamathiselwe):
•Khulisa izinga lokushisa le-annealing - zama ukwenza amabhondi e-hydrogen abuthakathaka angakwazi ukuwagcina;
·Fushanisa isikhathi sokukhipha i-anneal kanye ne-elongation - nciphisa ithuba lamabhondi e-hydrogen abuthakathaka;
·Yehlisa ukugxila kwe-primer - nciphisa amathuba okubopha ama-primer angasebenzi kanye nezifunda okungahlosiwe;
Ukusebenza kahle kwe-amplification
Isimo esiphambene nokukhulisa okungaqondile - ukusebenza kahle kokukhulisa usayizi okuphansi , kanye nezinyathelo zokubhekana nokusebenza kahle kokukhulisa okuphansi zihlukile nje:
•Yelula isikhathi sokukhipha kanye nokwelula;
·Shintshela ku-PCR enezinyathelo ezintathu futhi wehlise izinga lokushisa lokudonsa;
•Khulisa ukugxilisa ingqondo kuqala;
Ps: Abafundi abaningi abathweswe iziqu abazalwa ngeminyaka yama-90s abazimisele ukufunda indlela yokulungisa izivivinyo, futhi banethemba lokuthi ikhithi ingaxazulula inkinga ngokuphelele (uma ufuna ukuya enkampanini esebenza ngokusebenza kabusha ukuze wenze ucwaningo nentuthuko ngemuva kokuthweswa iziqu), empeleni, abakhiqizi be-reagent nabo bacabanga ngale ndlela, ngithemba ukuthi kuwubuwula Ingasetshenziswa uma uyitholile, ngakho-ke abakhiqizi be-reagent basebenzise umzamo omkhulu ekuxazululeni inkinga yokungafinyeleli ekuxazululeni inkinga ebuthakathaka. Izici zokumuncwa kwe-H-bond.Ukuxazulula inkinga kalula, iziwula kusafanele zifunde ukwethulwa kwenkampani ye-reagent ukuze zibone ukuthi sikhona yini isici esidonsa izibopho ze-hydrogen ezibuthakathaka.
Ukukhetha okunzima kokugxila kwe-primer kanye nenkinga ngama-primer-dimers
Indlela 1: Ngokuvamile, imiyalelo yekhithi ye-qPCR inamasistimu anconyiwe kanye nokugxilisa kokuqalisa okunconyiwe.
Indlela yesi-2: Ukulungisa iphutha ngokusetha i-primer concentration gradient.Isithombe esingezansi sebiwe enkampanini ukuze siboniswe.Isibalo esingezansi sibonisa imiphumela yobuningi be-fluorescence eyenziwe ngama-primer concentration gradients (100nM, 250nM, 500nM) kanye nama-gradients okugxiliswa kwezifanekiso ezine (0.1ng, 1ng, 10ng, 100ng).Inani le-Ct lemiphumela yokuhlola lihlelwe kanje:

I-Primer Concentration Selection Hlanganisa ukugxilisa kwesiqalo ngasinye emgqeni kanje:

Ukukhethwa kokugxiliswa kwe-primer kusobala, ubudlelwano bomugqa bokugxilwa kwe-primer engu-100nM kanye no-250nM bungcono, futhi ubudlelwano bomugqa bokugxila kokugxilwa kwe-primer engu-500nM bubi kakhulu.Ku-100nM naku-250nM, inani le-Ct lika-250nM lincane uma kuqhathaniswa, ngakho-ke ukugxilwa kwe-primer okungcono kakhulu ngu-250nM.Ngokuvamile ama-primer-dimers aqinile angabonakala ejikeni elincibilikayo.Kuthiwani uma ama-primer aklanyelwe engakwazi ukugwema ama-primer-dimers?
Indlela yesi-3: Yehlisa inani lama-primers futhi ukhuphule izinga lokushisa le-anneal (asikho isidingo sokuchaza).
Inani le-empirical lokushisa kwe-annealing lingu-60°C.Uma ungaqiniseki, ungakhetha kanjani izinga lokushisa le-anneal elifaneleka kakhulu?Impendulo iyafana nokukhetha kwe-primer concentration -ukuhlolwa kwe-gradient.Thatha isithombe enkampanini ye-Bio-rad ukukhombisa inkinga.Ukuze ukhulise ucezu oluthile oluqondiwe, setha ama-gradients okushisa ayisishiyagalombili, ngalinye libe nezimpinda ezintathu, futhi ijika lokukhulisa elitholiwe limi kanje:

Ukukhethwa kwezinga lokushisa le-annealing :
·70°C, 69°C—Eqinisweni, iziqalo azikwazi ukuhlanganiswa, ngakho akukho ukukhuliswa.
·67.3°C - Kukhona inani elincane lokukhulisa ekuqaleni, futhi inani le-Ct likhulu ngokuqhathaniswa.
·64.5°C——Inani le-Ct liyehla.
·Ku-60.7°C, 58.0°C, 56.2°C, kanye no-55.0°C, amanani e-Ct ngokuyisisekelo ayethambekele ekuzinzeni, kodwa amanani okugcina e-fluorescence ayehlukile.
Indlela yokukhetha?Isimiso: Isimiso sokuqala yinani eliphakeme le-Ct.Ngevelu efanayo ye-Ct, khetha izinga lokushisa eliphezulu lokuthungatha ukuze ugweme ukwenziwa kwe-dimerization kanye nokukhulisa okungaqondile.Nakuba kunevelu ephezulu ye-fluorescence engu-55°C, kungase kube nama-dimers noma ukukhulisa okungaqondile kuwo.
Kodwa uma uhlakaniphe njengawe, uzocabanga nakanjani: Uma ukhuluma ngokunengqondo, uma ukusabela kwe-PCR kucace kakhulu, inqobo nje uma ukugxilwa kwe-primer kudlula imfuneko encane, amaphuzu aphezulu naphansi akufanele abe nomphumela, njengodayi be-fluorescent kanye nama-dNTP.Ngempela, inqobo nje uma izinga lokushisa le-anneal lilungiselelwe kahle, umthelela we-primer concentration enanini le-Ct uzoncishiswa ngokwemvelo.

Izinga lokushisa le-anneal lilungiselelwe kahle, futhi umphumela wokugxilisa i-primer ku-CT uzoncishiswa
Isakhiwo sesibili sithinta ukusebenza kahle kokukhulisa
Ake sithathe isithombe ku-Bio-rad ukukhombisa inkinga.Iphinde idizayine i-gradient yezinga lokushisa ukuze ikhulise isakhi sofuzo ngesakhiwo sesibili.

Isakhiwo sesibili siyavela
Kungabonakala ukuthi njengoba izinga lokushisa lincipha, imikhiqizo iqala ukubonakala futhi inani le-Ct liqhubekela phambili, lifinyelela inani elincane ku-60.7 ° C, bese kuthi lapho izinga lokushisa liyancipha, inani le-Ct liba likhulu.Ngokuphambene, njengoba izinga lokushisa likhuphuka, isakhiwo sesibili siyavuleka futhi ukusebenza kahle kokukhulisa kuyanda.Ngemva kokufinyelela izinga elithile lokushisa, ukwandisa izinga lokushisa akukwazi ukuthuthukisa ukusebenza kahle kokukhulisa.Ngoba ama-primers awakwazi ukuhlanganiswa ngokuzinza ngalesi sikhathi.Ngakho-ke,bheka izinga lokushisa elinenani eliphansi le-Ct, okuyizinga lokushisa elingcono kakhulu lokukhulisa isifanekiso sesakhiwo sesibili!Yiqiniso, iziwula ezihlakaniphile kufanele zazi ukuthi uma kungenasidingo, kungcono ukushintsha ama-primers futhi ugweme isifunda sesakhiwo sesibili.
5. Izinga lesicelo
I-MIQE—Ukuhlaziywa Kwedatha

Ukuhlaziywa kwedatha kunikezwa ikakhulukazi ithuluzi le-PCR le-fluorescent quantitative.Esihlokweni esandulele, umsebenzi omningi wokuhlaziya idatha wenziwa, njengokulawula okungenalutho, okuye kwachazwa ekwakhiweni kokuhlolwa.Izakhi zofuzo eziyisethenjwa zangaphakathi, izinombolo eziphindayo, njll. ziye zacaciswa., lapha sichaza ikakhulukazi ukusetshenziswa kwe-qPCR.
I-qPCR isetshenziswa kabanzi, futhi ukuqinisekiswa kokuhlolwa kanye nokuxilongwa kwe-nucleic acid yizimo ezisetshenziswa kakhulu.
i-quantification ephelele
Ilogi (ukugxila kokuqala) inobudlelwano bomugqa nenani lemijikelezo.Ijika elijwayelekile lingathathwa kusukela kwendinganiso enenombolo yekhophi yokuqala eyaziwayo, okungukuthi, ubudlelwano bomugqa bokusabela kokukhulisa kungatholwa.Ngokwevelu ye-Ct yesampula, ukugxila kusampula kungabalwa.Inani lezifanekiso ezizofakwa.

Indlela Yekubala Yesilinganiso Esiphelele
Ukulinganisa okuphelele kufanele kusekelwe kwijika elijwayelekile.Ukwenza ijika elijwayelekile, kudingeka izinga.Ngokuvamile, indinganiso iyi-plasmid etholakala ngokuhlanganisa isakhi sofuzo esiqondiwe.Kungani i-plasmid?Ngoba i-plasmid DNA eyindilinga iyona ezinzile kakhulu.Nciphisa umkhiqizo ojwayelekile ngama-gradients angu-5 ukuya kwangu-6 ngokwesilinganiso esiphindwe kabili (i-dilution ephindwe ka-10), futhi unake ukufana lapho uhlanza.Vumela inani le-Ct lehle phakathi kuka-15-30.

Ukulungiselela okujwayelekile
Ngesikhathi esifanayo, isampula okufanele ihlolwe kufanele futhi ihlanjululwe ngokufanele (khumbula i-dilution factor), futhi inani le-Ct kufanele futhi lehle phakathi kuka-15-30.Umkhiqizo ojwayelekile + isampuli okufanele ihlolwe ifakwa emshinini ndawonye.Ngemva kokugijima, ijika elijwayelekile lenziwa ngento evamile, futhi amasampuli azohlolwa alethwa ejikeni elijwayelekile ukuze kubalwe ukugxilisa ingqondo.
I-Hepatitis B virus I-HBV quantification ukulinganisa okuphelele okujwayelekile, okungabala inombolo yekhophi yegciwane ku-1ml yegazi.
Ukubalwa kwenombolo yekhophi
Ukuhlushwa kwesampula okufanele kuhlolwe (ng/ul) = OD260 × 50ug/ml × isici sokuhlanjululwa
Isampula lesisindo samangqamuzana = inani lezisekelo × 324
Inombolo yekhophi yesampula ezohlolwa (amakhophi/ul) = ukugxiliswa kwesampula okufanele ihlolwe / isisindo samangqamuzana esampula × 6 × 1014

Indlela yokubala inombolo yokukopisha

Okungenhla kuyindlela yokubala yokunquma inani.Lena inkinga yezibalo engaxazululeka ngemva kokuphothula esikoleni esiphakeme, futhi izinkinga zezibalo ngokuvamile zixazululwa ngamakhompyutha.Uma ungaqondi, ungeza ukuze uxhumane.
isilinganiso esihlobene
Ukulinganisa okuhlobene kusetshenziswa kakhulu ocwaningweni lwesayensi.Mangaki amagciwane akhona ku-1ml wegazi, futhi igciwane le-DNA, lesi yisenzakalo esinqunyiwe: inani legazi linganqunywa, futhi igciwane le-DNA lizinzile.Kodwa-ke, kunzima ngathi ukuqhathanisa inani lamakhophi alotshiweyo wofuzo oluthile eqabungeni, ngoba kunzima ukunquma usayizi, isisindo, nokuthamba kweqabunga, inani le-RNA ekhishiwe kunzima ukulinquma, kanye nokusebenza kahle kokulotshwa okuhlanekezelwe nakho kunzima ukunquma, okusho ukuthi, noma yisiphi isinyathelo singenza idatha yokuhlola ibe nezimbungulu futhi ayikwazi ukusetshenziswa.
Ngakho-ke, ubuningi obuhlobene kufanele bethule isici:isakhi sofuzo esibhekisela ngaphakathi .
Ngamanye amazwi, ukulinganisa okuhlobene empeleni kuwukuqhathanisa phakathi kofuzo oluqondiwe kanye nofuzo lwangaphakathi lwereferensi.Uma kuqhathaniswa kusicubu esifanayo neseli efanayo, umthelela wosayizi wesampula, inani lokukhishwa kwe-RNA, ukusebenza kahle kokuhlehla kokuloba, nokusebenza kahle kwe-PCR kuncane uma kuqhathaniswa.Ngenxa yosayizi omncane wesampula, kokubili izakhi zofuzo zenkomba zangaphakathi kanye nezakhi zofuzo ezihlosiwe zehlisiwe ngokuqhathaniswa.Yingakho kade sigcizelela ukufana nokuzinza phambilini.
Izakhi zofuzo eziyisethenjwa zangaphakathi zijwayelekileizakhi zofuzo( izakhi zofuzo ezigcina indlu), ezibhekisela esigabeni sezakhi zofuzo ezivezwa ngokuzinzile kuwo wonke amangqamuzana, futhi imikhiqizo yawo iyadingeka ukuze kugcinwe imisebenzi eyisisekelo yokuphila kwamaseli.
Ungawuphambanisi lo mqondo.Izakhi zofuzo zokugcinwa kwezindlu zingamagama omsebenzi webhayoloji, kanti ufuzo lwangaphakathi oluyisethenjwa lungamagama obuchwepheshe okuhlola.Izakhi zofuzo zokugcina izindlu zidinga ukudlula ukuqinisekiswa ngaphambi kokuthi zikhethwe njengezakhi zofuzo eziyisethenjwa zangaphakathi.
Isibonelo, sikhethe izakhi zofuzo ezimbalwa zokugcina indlu emfanekisweni ongezansi ukuze sihlole amazinga azo okukhuluma kumaseli ezicubu ezihlukene, futhi sathola ukuthi amazinga enkulumo ye-β-2-microglobulin ayehluke kakhulu kulawo amanye amathathu ofuzo, ngakho awakwazanga ukusetshenziswa njengofuzo lwangaphakathi lwereferensi.

Ngemva kokuqonda umsebenzi wokulungisa wofuzo lwangaphakathi lwereferensi, ama-algorithms amabili atholakala ngenxa yokwethulwa kofuzo lwangaphakathi lwereferensi.
·indlela yejika elijwayelekile elikabili
·2 – △△Ct indlela (indlela yokuqhathanisa inani le-CT)
Uma ungathanda ukufunda izinhlobo nemisebenzi yofuzo, sicela uyeke ucwaningo lwama-algorithms futhi usebenzise amafomula ngokuqondile, noma usebenzise imishini ngokuqondile;uma ungumuntu oqondile kwizibalo nobunjiniyela, sicela ukhululeke.
indlela yejika ephindwe kabili
Linganisa ufuzo oluqondiwe kanye nofuzo lokugcina indlu lwesampula yokulawula kanye nesampula okufanele ihlolwe ngejika elijwayelekile, bese ubala inani elihlobene ngokuya ngefomula yokubala, okuyileveli yesisho esihlobene.
Izinzuzo: ukuhlaziya okulula, ukwenza isilingo esilula
Ububi: Kufuzo ngalunye, umjikelezo ngamunye wokuhlolwa kufanele wenze ijika elijwayelekile
Isicelo: Enye yezindlela ezimbili ezivame ukusetshenziswa kakhulu neziqashelwayo zokulinganisa isihlobo esifundweni sokulawulwa kokuvezwa kofuzo
Ifomula imi kanje:

Izibonelo zimi kanje:

Bala inani elihlobene ngokusekelwe kumphumela wobuningi
2 – △△Ct indlela (indlela yokuqhathanisa inani le-CT)

Izinzuzo: Asikho isidingo sokwenza ijika elijwayelekile
Ukungalungi: Kucatshangwa ukuthi ukusebenza kahle kokukhulisa kusondele ku-100%;ukuchezuka okujwayelekile kungu-< 5%, futhi ijika elijwayelekile kanye nokusebenza kahle phakathi kokukhulisa ngakunye kuthathwa njengokufana;ukwenziwa kahle kwezimo zokuhlola kuyinkimbinkimbi kakhulu.
Isicelo: Enye yezindlela ezimbili ezivame ukusetshenziswa kakhulu neziqashelwayo zokulinganisa isihlobo esifundweni sokulawulwa kokuvezwa kofuzo

Yebo, ukusebenza kahle kokukhulisa ngokuvamile akwenzeki ukuthi kube 1. Indlela yokulungisa: Uma sazi ukuthi isakhi sofuzo esiqondiwe kanye nofuzo lwereferensi lunokusebenza kahle kokukhulisa usayizi, kodwa ukusebenza kahle kokukhulisa akulingani no-1, khona-ke u-2△△Ct angalungiswa njengokuthi: (1+E )－△△ukwenziwa kahle kwe-Ct, isibonelo, i-Ct, isibonelo, i-amplified 0, isibonelo, ingakwazi ukulungiswa kahle. 1.95－△△Ct
Kuze kube manje, okuqukethwe mayelana ne-fluorescent quantitative PCR sekufike esiphethweni .