Ukucaciswa kokutholwa

Ezimweni eziningi, inhloso ye-primer design ukukhulisa ukucaciswa kwe-PCR.Lokhu kunqunywa umthelela omningi noma omncane ongabikezelwa wezinhlobonhlobo eziningi.Okuhlukile okukodwa okubalulekile ukulandelana okusekugcineni kuka-3′ kwesiqalo.

Okubalulekile, izivivinyo ze-PCR eziklanyelwe ukucacisa maningi amathuba okuthi zigcine ukusebenza kahle okuphezulu phezu kwebanga elibanzi eliguquguqukayo, ngoba ukulinganisa akukhiqizi imikhiqizo yokukhulisa engaqondile, ngaleyo ndlela kuncintisana nama-reagents e-PCR noma kuvimbele ukusabela okuyinhloko kokukhulisa.

Yiqiniso, kwezinye izimo, ukucaciswa akuyona into ebaluleke kakhulu, isibonelo, lapho umgomo uwukulinganisa amagciwane ahlobene eduze kodwa ahlukene, umklamo okhethekile, ukuthuthukiswa kanye namazinga okuqinisekisa ayadingeka.

Ijika elincibilikayo liyindlela evamile yokuhlola ukucaciswa kwama-amplicons, okungenani mayelana nokuthi kunwetshwe yini okuhlosiwe okukodwa.Kodwa-ke, kufanele kugcizelelwe ukuthi amajika ancibilikayo angadukisa ngoba, isibonelo, angathinteka emiphumeleni ehlanganisiwe yama-primers aphansi kanye nokugxila okuphansi kwesifanekiso.

P5 |Ijika elincibilikayo libonisa ukushintshwa kwe-Tm okutholwe ekutholweni okubili kwamanani ahlukene ama-DNA amabili okuhlosiwe.

A. Ekugxiliseni okuphezulu (isikhangiso)), ayikho i-primer dimer esobala ngemva kokuqedwa kokulinganisa kwe-qPCR.Njengoba ukugxiliswa kwesifanekiso kuncipha kuya kumakhophi angu-50 (e), umkhiqizo ongaqondile uqala ukuvela futhi uba ukuphela komkhiqizo ekugxiliseni okuphansi kakhulu (f).

B. Ukuhlolwa kuqophe ama-Tms afanayo kukho konke ukugxilisa okuqondiwe, futhi akuzange kube khona i-primer dimer esobala ngisho nasezindaweni eziphansi kakhulu (amakhophi angu-5).Uma usebenzisa lezi zindlela zokuthola ezimbili, ayikho imikhiqizo yokukhulisa etholwe kuma-NTC.

I-P5 ikhombisa amajika okuhlakazeka atholwe ngamasampula lapho isifanekiso sikhona ngokugxila okuhlukile.I-P 5a ibonisa ukuthi ekugxilweni okubili okuphansi kakhulu, i-Tms yemikhiqizo yokukhulisa engaqondile ekhiqizwayo iphansi kunaleyo yama-amplicon athile.

Ngokusobala, le ndlela yokuthola ayikwazi ukusetshenziselwa ukuthola okuhlosiwe okukhona ezindaweni eziphansi.

Kuyathakazelisa ukuthi ama-NTC, okungukuthi, amasampula angenayo i-DNA nhlobo, awazange arekhode (okungaqondile) imikhiqizo yokukhulisa i-genomic, okubonisa ukuthi i-DNA ye-genomic yangemuva ingabamba iqhaza ekukhuliseni/kupholiseni okungaqondile.

Kwesinye isikhathi ama-primer angemuva anjalo nokukhulisa okungaqondile akukwazi ukulungiseka, kodwa kuvame ukukwazi ukuklama indlela yokuthola engenakho ukukhulisa okungaqondile kunoma yikuphi ukugxiliswa kwesifanekiso kanye ne-NTC (P 5b).

Lapha, ngisho nokurekhoda ukukhuliswa kokugxiliswa okuhlosiwe nge-Cq engu-35 kuzoveza ijika elithile lokuhlakazeka.Ngokufanayo, ama-NTC awazange abonise izimpawu zokukhuliswa okungaqondile.Kwesinye isikhathi, indlela yokuthola ingase incike otshwaleni kamama, futhi kutholwa ukukhulisa okungaqondile kuphela ekwakhiweni okuthile kwebhafa, okungase kuhlobane nokugxilisa okuhlukile kwe-Mg2+.

Ukuzinza kokutholwa

Ukulungiselelwa kwe-Ta kuyisinyathelo esiwusizo ekuqinisekiseni okunamandla kanye nenqubo yokuthuthukisa yokutholwa kwe-qPCR.Inikeza inkomba eqondile yokuqina kwe-primer ebekwe ngokubonisa izinga lokushisa (noma izinga lokushisa) elikhiqiza i-Cq ephansi kakhulu ngaphandle kokukhulisa i-NTC.

Umehluko ophindwe kabili kuya kane ekuzweleni ungase ungabaluleki kubantu abanenkulumo ephezulu ye-mRNA, kodwa ezivivinyweni zokuxilonga, kungase kusho umehluko phakathi kwemiphumela engalungile nengalungile.

Izakhiwo ze-Ta ze-qPCR primers zingahluka kakhulu.Okunye ukuhlola akuqinile kakhulu, futhi uma kungenziwanga ngaphansi kwenani elifanele lika-Ta lama-primers, zizowa ngokushesha.

Lokhu kubalulekile ngoba lolu hlobo lokutholwa luvamise ukuba yinkinga emhlabeni wangempela, futhi ukuhlanzeka kwesampula, ukugcwala kwe-DNA, noma ukuba khona kwenye i-DNA kungase kungabi kuhle.

Ngaphezu kwalokho, inombolo yekhophi eqondiwe ingase ihluke ebangeni elibanzi, futhi ama-reagents, izitsha zepulasitiki, noma amathuluzi angase ahluke kulezo ezisetshenziswa lapho kusethwa isivivinyo.

P6|Izinga lokushisa libonisa ukuqina okuhlukile kokutholwa kwe-PCR.

A. Sebenzisa i-Bioline's Sensifast SYBR mastermix (inombolo yekhathalogi engu-BIO-98050) ukwenza i-PCR ku-cDNA elungiswe nge-RNA yobuchopho bomuntu.

B. Sebenzisa ithuluzi le-CFX qPCR le-Bio-Rad ukurekhoda imephu yokukhulisa izwi kanye nejika lokuqedwa le-apalene (NM_033207, F: GCCATGGAGGAAAGTGACAGACC, R: CTCATGTGTGGGTGATCTCCTAGG).

C. Igrafu yokukhulisa kanye nejika elincibilikayo le-ACSBG1 (NM_015162.4, F: CTACACTTCCGGCACCACTGG, R: GTCCACGTGATATTGTCTTGACTCAG).

D. Igrafu yokukhulisa kanye nejika lokuqedwa le-GFAP (NM_002055.5, F: TGGAGAGGAAGATTGAGTCGCTGG, R: CGAACCTCCTCCTCGTGGATCTTC).

I-E. Cqs irekhodwe ngamazinga okushisa ahlukene, ebonisa umehluko ku-Cq eqoshwe ngaphansi kwezinga lokushisa elingu-7C.

I-P 6 ibonisa umphumela ojwayelekile wokuhlolwa okungafuneki, lapho i-qPCR yenziwe khona kusetshenziswa i-gradient Tas phakathi kuka-59C no-67C (P 6a), kusetshenziswa ama-primer ezakhi zofuzo ezintathu eziqondene nobuchopho bomuntu.

Kungabonakala kugrafu yokukhulisa i-Opalin ukuthi ama-primers we-Opalin awalungile neze ngoba uhla lwawo olulungile lwe-Ta luncane kakhulu (Umfanekiso 6b), okungukuthi, ama-Cq ahlakazeke kakhulu, okuholela ekutheni ama-Cq aqhathaniswe ngokuphawulekayo nama-Cqs awo aphansi.

Le ndlela yokuthola ayizinzile futhi ingase iholele ekukhuliseni okungaphansi.Ngakho-ke, le pair of primers kufanele iklanywe kabusha.Ngaphezu kwalokho, ukuhlaziywa kwejika elincibilikayo (okufakiwe) kubonisa ukuthi ukucaciswa kwale ndlela yokuthola kungase kube yinkinga, ngoba ijika lokuncibilika le-Ta ngayinye lihlukile.

Indlela yokuthola i-ACSBG1 eboniswe ku-P 6c iqine kakhulu kunendlela yokuthola i-Opalin ngenhla, kodwa isekude kakhulu nokuhle, futhi kungenzeka ukuthi ingathuthukiswa.

Kodwa-ke, sigcizelela ukuthi akukho ukuxhumana okudingekile phakathi kokuqina nokucacisa, ngoba ijika lokuhlakaza elikhiqizwa yile ndlela yokuthola libonisa inani eliphakeme elifanayo kuwo wonke ama-Tas (okufakwayo).

Ngakolunye uhlangothi, ukuhlolwa kokuqina kuyabekezelela kakhulu, kukhiqiza ama-Cq afanayo ku-Tas anhlobonhlobo, njengoba kuhlolo lwe-GFAP oluboniswe ku-P 6d.

Umehluko kuma-Cqs atholwe ebangeni elifanayo elingu-8 degrees Celsius ungaphansi kuka-1, futhi ijika lokuhlakaza (okufakwa kulo) liqinisekisa izici zokutholwa kuleli banga lokushisa.Kuyaphawuleka ukuthi i-Tas ebaliwe kanye nobubanzi bangempela be-Ta kungase kuhluke kakhulu.

Kuneziqondiso eziningi eziklanyelwe ukusiza abacwaningi ukuba bakhe ama-primers asebenza kahle, amaningi awo asekelwe emithethweni yakudala yasungulwa futhi ukunakwa okuningi kukhokhwe ku-3′end of the primers.Kuvame ukutuswa ukuthi kufakwe u-G noma u-C ekugcineni okungu-3' kanye nezisekelo ezimbili ze-G noma ze-C (i-GC clamp), kodwa zingadluli izisekelo ezimbili kwezingu-5 zokugcina.

Empeleni, le mithetho ingaqondisa abacwaningi, kodwa ayilungile ngaphansi kwazo zonke izimo.

P7 |I-3′end ye-primer inomthelela omncane ekucaciseni noma ekusebenzeni kahle.

A. Isikhundla seziqalisi zofuzo lwe-HIF-1α (NM_181054.2) yomuntu.

B. Sebenzisa i-Agilent Brilliant III SYBR Utshwala kamama obuluhlaza (Cat No. 600882) ukuze ukhulise izinto zokuhlola eziyisithupha.

C. Igrafu yokukhulisa kanye nejika elincibilikayo elirekhodwe ithuluzi le-CFX qPCR le-Bio-Rad kanye nama-primer angu-3′end.Ama-NTC aboniswa ngokubomvu.

D. Cqs irekhodi lento ngayinye yokuhlola

Isibonelo, umphumela ku-P 7 uphikisana nomthetho we-3′end.Yonke imiklamo ikhiqiza imiphumela efanayo, enezinhlanganisela ezimbili kuphela eziholela ekukhuliseni okungaqondile ku-NTC.

Kodwa-ke, asikwazi ukusekela umthelela wesiqeshana se-GC, ngoba kulesi simo, ukusebenzisa u-A noma u-T njengomkhawulo wezisekelo ezingama-30 akunciphisi ukucaciswa.

Isivivinyo C, lapho i-primer ka-F igcina khona ngo-GGCC, irekhodile ama-Cqs kuma-NTC, okubonisa ukuthi umuntu angase afune ukugwema lokhu kulandelana ekupheleni kuka-30.Sigcizelela ukuthi okuwukuphela kwendlela yokunquma ukulandelana okungcono kakhulu kwe-3′ kwepheya yokuqala ukuhlola ama-primers athile ngokuhlolwa.

Ukusebenza kahle kokukhulisa

Okubalulekile, nakuba ukutholwa kwe-PCR okungaqondile akusoze kwacaciswa, ukusebenza kahle kokukhulisa kungalungiswa futhi kukhuliswe ngezindlela eziningi ezihlukene ngokushintsha i-enzyme, utshwala bomama, izithasiselo, nezimo zamabhayisikili.

Ukuze uhlole ukusebenza kahle kokutholwa kwe-PCR, kungcono kakhulu ukusebenzisa i-serial dilution ye-10 noma izikhathi ezi-5 kune-nucleic acid ehlosiwe, okungukuthi, "indlela yejika evamile".

Uma ama-amplicon e-PCR noma okuqondiwe kwe-DNA yokwenziwa kusetshenziswa ukukhiqiza ijika elijwayelekile, ukuhlanjululwa okulandelanayo kwalokhu okuhlosiwe kufanele kuhlanganiswe nenani elingaguquki le-DNA yangemuva (njenge-genomic DNA).

P8 |Ijika le-Dilution lokuhlola ukusebenza kahle kwe-PCR.

A. Sebenzisa okokuqala kwe-HIF-1: F: AAGAACTTTTAGGCCGCTCA kanye ne-R: TGTCCTGTGGTGACTTGTCC ne-Agilent's Brilliant III SYBR Green mastermix (inombolo yekhathalogi 600882) ye-PCR nezimo zejika elincibilikayo.

B. I-RNA eyi-100 ng yabhalwa kabusha, yahlanjululwa izikhathi ezi-2, futhi amasampula e-cDNA ahlanjululwe uchungechunge ahlanjululwa izikhathi ezi-5 kuya ku-1 ng i-DNA yomuntu.Ijika elincibilikayo liboniswa esihlokweni.

C. Ukusabela kwe-RT, ukuhlanjululwa, kanye nokuhlanjululwa kwe-serial kwaphindwa kusampula yesibili ye-cDNA, futhi imiphumela yafana.

I-P 8 ibonisa amajika amabili ajwayelekile, kusetshenziswa indlela efanayo yokuthola kumasampuli amabili ahlukene e-cDNA, umphumela uwukusebenza kahle okufanayo, cishe u-100%, futhi inani le-R2 nalo liyafana, okungukuthi, izinga lokulingana phakathi kwedatha yokuhlola kanye nomugqa wokuhlehla noma idatha I-Degree of linearity.

Amajika amabili ajwayelekile ayaqhathaniswa, kodwa awafani ncamashi.Uma inhloso kuwukubala kahle ithagethi, kufanele kuqashelwe ukuthi akwamukelekile ukunikeza ikhophi inombolo yokubala ngaphandle kokuchaza ukungaqiniseki.

P9 |Ukungaqiniseki kokulinganisa okuhlobene nokulinganisa kusetshenziswa ijika elijwayelekile.

A. Sebenzisa okokuqala kwe-GAPDH (NM_002046) ukwenza i-PCR nezimo zejika lokuncibilika.F: ACAGTTGCCATGTAGACC kanye ne-R: TAACTGGTTGAGCACAGG kanye ne-Bioline's Sensifast SYBR mastermix (inombolo yekhathalogi engu-BIO-98050).

B. Ishadi lokukhulisa, ijika elincibilikayo kanye nejika elijwayelekile elirekhodwe ngethuluzi le-CFX qPCR le-Bio-Rad.

C. Igrafu yejika elijwayelekile kanye nesikhawu sokuzethemba esingu-95% (CI).

D. Inombolo yekhophi kanye nesikhawu sokuzethemba esingu-95% samanani amathathu e-Cq asuselwe kwijika lokuhlanjululwa.

I-P 9 ibonisa ukuthi ekuhlolweni okuthuthukisiwe, ukuhlukahluka okungokwemvelo kwejika elilodwa elijwayelekile cishe izikhathi ezingu-2 (isikhawu sokuzithemba esingu-95%, ubuncane kuya kokuningi), okungahle kube ukuhluka okuncane okungalindelwa.

Umkhiqizo ohlobene:

I-Cell Direct RT qPCR Kit

Ikhithi ye-Mouse Tail Direct PCR

Ikhithi ye-PCR ye-Animal Tissue Direct