I-RT-qPCR ithuthukiswe kusukela kubuchwepheshe obujwayelekile be-PCR.Yengeza amakhemikhali e-fluorescent (odayi be-fluorescent noma ama-fluorescent probes) kusistimu yokusabela ye-PCR evamile, futhi ithola inqubo yokukhishwa kwe-PCR nesandiso ngesikhathi sangempela ngokuya ngezinqubo zabo zokukhanya ezihlukile.Izinguquko zesignali ye-fluorescent phakathi nendawo zisetshenziselwa ukubala inani loshintsho lomkhiqizo kumjikelezo ngamunye we-PCR.Njengamanje, izindlela ezivame kakhulu indlela yokudaya ye-fluorescent kanye nendlela yokuhlola.

Indlela yokudaya ye-Fluorescent:
Abanye odayi be-fluorescent, njenge-SYBR Green Ⅰ, i-PicoGreen, i-BEBO, njll., abakhiphi ukukhanya ngokwabo, kodwa bakhipha i-fluorescence ngemva kokubophezela ku-groove encane ye-dsDNA.Ngakho-ke, ekuqaleni kokusabela kwe-PCR, umshini awukwazi ukubona isignali ye-fluorescent.Lapho ukusabela kuqhubeka ku-annealing-extension (indlela yezinyathelo ezimbili) noma isigaba sokunwetshwa (indlela yezinyathelo ezintathu), izintambo ezikabili ziyavulwa ngalesi sikhathi, kanye ne-DNA polymerase entsha Ngesikhathi sokuhlanganiswa kwe-strand, ama-molecule e-fluorescent ahlanganiswa ku-dsDNA encane groove futhi akhiphe i-fluorescence.Njengoba inani lemijikelezo ye-PCR likhula, odayi abaningi ngokwengeziwe bayahlangana ne-dsDNA, futhi isignali ye-fluorescent nayo ithuthukiswa ngokuqhubekayo.Thatha i-SYBR Green Ⅰ njengesibonelo.
Indlela yokuhlola:
I-Taqman probe iwuphenyo oluvame ukusetshenziswa kakhulu lwe-hydrolysis.Kukhona iqembu le-fluorescent ekugcineni kuka-5′ we-probe, ngokuvamile i-FAM.I-probe ngokwayo iwukulandelana okuhambisanayo nofuzo oluqondiwe.Kukhona iqembu lokucisha i-fluorescent ekugcineni kuka-3′ we-fluorophore.Ngokomgomo we-fluorescence resonance energy transfer (Förster resonance energy transfer, FRET), lapho iqembu lentatheli ye-fluorescent (donor fluorescent molecule) kanye neqembu le-fluorescent elicishayo (i-acceptor fluorescent molecule) Lapho i-spectrum ejabulisayo igqagqana futhi nebanga liseduze kakhulu (7-10nm ikhipha 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.

Fig 1 Isimiso se-qRT-PCR

Idizayini yokuqala
Izimiso:

Ama-primers kufanele aklanywe endaweni elondoloziwe yochungechunge lwe-nucleic acid futhi abe nokucacile.

Kungcono ukusebenzisa ukulandelana kwe-cDNA, futhi ukulandelana kwe-mRNA nakho kuyamukeleka.Uma kungenjalo, thola idizayini yesifunda yama-cd yokulandelana kwe-DNA.
Ubude bomkhiqizo we-fluorescent quantitative ngu-80-150bp, omude kakhulu ngu-300bp, ubude be-primer ngokuvamile buphakathi kwezisekelo ezingu-17-25, futhi umehluko phakathi kweziqalo ezikhuphuka nomfula akumele zibe mkhulu kakhulu.

Okuqukethwe kwe-G+C kuphakathi kuka-40% no-60%, kanti u-45-55% uhamba phambili.
Inani le-TM liphakathi kuka-58-62 degrees.
Zama ukugwema ama-primer dimers nama-self-dimers, (angaveli ngaphezu kwamapheya angu-4 ezisekelo ezihambisanayo ezilandelanayo) isakhiwo se-hairpin, uma kungenakugwema, yenza i-ΔG<4.5kJ/mol* Uma ungakwazi ukuqinisekisa ukuthi i-gDNA isusiwe ngesikhathi sokuloba okuhlanekezelwe Hlanza, kungcono ukudizayina ama-primers we-intron, C. isakhiwo esiqhubekayo (2-3) iziqalo nezingezona
ethize I-homology yokulandelana kwe-amplified heterogeneously ingaphansi kwama-70% noma ine-homology eyisisekelo eyisi-8 ehambisanayo.
Isizindalwazi:
Usesho lwe-CottonFGD ngamagama angukhiye
Idizayini yokuqala:
Idizayini yokuqala ye-IDT-qPCR

Ikhasi lethuluzi lethuluzi lokuklama le-Fig2 IDT eliku-inthanethi

Isibonisi sekhasi lemiphumela ye-Fig3
Idizayini yeziqalo ze-lncRNA:
I-lncRNA:izinyathelo ezifanayo njenge-mRNA.
i-miRNA:Umgomo wendlela ye-stem-loop: Njengoba wonke ama-miRNA angukulandelana okufushane okungaba ngu-23 nt, ukutholwa kwe-PCR okuqondile akukwazi ukwenziwa, ngakho kusetshenziswa ithuluzi lokulandelana kwe-stem-loop.Ukulandelana kwe-stem-loop kuyi-DNA enomucu owodwa engaba ngu-50 nt, engakha isakhiwo se-hairpin ngokwayo.3 'Isiphetho singaklanywa njengokulandelana okuhambisana nengxenye yengxenye ye-miRNA, bese i-miRNA eqondiwe ingaxhunywa ngokulandelana kwe-stem-loop ngesikhathi sokuloba okuphambene, futhi ubude obuphelele bungafinyelela ku-70bp, obuhambisana nobude bomkhiqizo okhulisiwe obunqunywe yi-qPCR.Idizayini yokuqala ye-miRNA eyi-Tailing.
Ukutholwa kwe-Amplification-specific:
Isizindalwazi sokuqhuma kwe-inthanethi: I-CottonFGD iqhuma ngokulandelana kokufana
Ukuqhuma kwendawo: Bheka ekusebenziseni i-Blast+ ukwenza ukuqhuma kwasendaweni, i-linux nama-macos angasungula ngokuqondile isizindalwazi sendawo, uhlelo lwe-win10 lungenziwa ngemva kokufaka ubuntu bash.Dala idatha yendawo yokuqhuma kanye nokuqhuma kwendawo;vula ubuntu bash ku-win10.
Isaziso: Ukotini wase-Upland kanye nokotini wasesiqhingini sasolwandle kuyizitshalo ze-tetraploid, ngakho umphumela wokuqhuma ngokuvamile uzoba ukufana okubili noma ngaphezulu.Esikhathini esedlule, ukusebenzisa ama-cd e-NAU njengesizindalwazi ukwenza ukuqhuma kungenzeka kuthole izakhi zofuzo ezimbili ezilinganayo ezinomehluko ambalwa we-SNP.Ngokuvamile, izakhi zofuzo ezimbili ze-homologous azikwazi ukuhlukaniswa nge-primer design, ngakho-ke ziphathwa ngokufanayo.Uma kukhona i-indel ecacile, i-primer ivame ukuklanywa ku-indel, kodwa lokhu kungase kuholele esakhiweni sesibili se-primer Amandla amahhala abe ngaphezulu, okuholela ekunciphiseni kokusebenza kahle kokukhulisa, kodwa lokhu akunakugwenywa.

Ukutholwa kwesakhiwo sesibili sokuqala:
Izinyathelo:vula i-oligo 7 → ukulandelana kwesifanekiso sokufaka → vala iwindi elincane → gcina → thola i-primer kusifanekiso, cindezela u-ctrl+D ukuze usethe ubude be-primer → hlaziya izakhiwo zesibili ezihlukahlukene, ezifana nomzimba ozishintshayo, i-heterodimer, i-hairpin, ukungafani, njll. Izithombe ezimbili zokugcina kuMfanekiso 4 ziyimiphumela yokuhlola yeziqalo.Umphumela we-primer yangaphambili muhle, akukho sakhiwo esicacile se-dimer ne-hairpin, azikho izisekelo ezihambisanayo eziqhubekayo, futhi inani eliphelele lamandla wamahhala lingaphansi kuka-4.5, kuyilapho i-back primer ibonisa okuqhubekayo Izisekelo ezingu-6 ziyahambisana, futhi amandla amahhala angu-8.8;ngaphezu kwalokho, i-dimer engathi sína kakhulu ibonakala ekugcineni kwe-3, futhi i-dimer yezisekelo ezi-4 ezilandelanayo ibonakala.Nakuba amandla amahhala engephezulu, i-3′ dimer Chl ingathinta kakhulu ukucaciswa kokukhulisa kanye nokusebenza kahle kokukhulisa.Ngaphezu kwalokho, kuyadingeka ukuhlola ama-hairpins, ama-heterodimers, nokungahambisani kahle.

Fig3 oligo7 imiphumela yokuthola
Ukutholwa kokusebenza kahle kokukhulisa:
Ukusebenza kahle kokukhulisa ukusabela kwe-PCR kuthinta kakhulu imiphumela ye-PCR.Futhi ku-qRT-PCR, ukusebenza kahle kokukhulisa kubaluleke kakhulu emiphumeleni yobuningi.Susa ezinye izinto, imishini namaphrothokholi kusigcinalwazi sokusabela.Izinga lama-primers liphinde libe nomthelela omkhulu ekusebenzeni kahle kokukhulisa i-qRT-PCR.Ukuze kuqinisekiswe ukunemba kwemiphumela, kokubili ukulinganisa kwe-fluorescence okuhlobene kanye ne-absolute fluorescence quantification kudinga ukubona ukusebenza kahle kokukhulisa iziqalo.Kuyaqashelwa ukuthi Ukusebenza kahle kokukhulisa i-qRT-PCR kuphakathi kuka-85% no-115%.Kunezindlela ezimbili:
1. Indlela yejika evamile:
a.Hlanganisa i-cDNA
b.Ukwehliswa kwe-gradient
c.qPCR
d.Isibalo sokuhlehla komugqa ukuze kubalwe ukusebenza kahle kokukhulisa
2. I-LinRegPCR
I-LinRegPCR iwuhlelo lokuhlaziya Idatha ye-RT-PCR yesikhathi sangempela, ebizwa nangokuthi idatha ye-quantitative PCR (qPCR) esuselwe ku-SYBR Green noma kukhemistri efanayo.Uhlelo lusebenzisa idatha elungisiwe okungeyona eyesisekelo, lenza ukulungisa okuyisisekelo kusampula ngayinye Ngokuhlukana, inquma iwindi-of-linearity bese isebenzisa ukuhlaziywa kokuhlehla komugqa ukuze kulingane umugqa oqondile ngesethi yedatha ye-PCR.Kusukela emthambekeni walo mugqa ukusebenza kahle kwe-PCR kwesampula ngayinye kuyabalwa.Isilinganiso se-PCR esisebenza kahle nge-amplicon ngayinye kanye nenani le-Ct ngesampuli ngayinye kusetshenziselwa ukubala ukugxilisana kokuqala ngesampula ngayinye, evezwa ngamayunithi e-fluorescence angaqondakali.Ukufakwa kwedatha nokuphumayo kungesipredishithi se-Excel.Isampula kuphela
ukuxuba kuyadingeka, akukho gradient
kudingeka izinyathelo:(Thatha i-Bole CFX96 njengesibonelo, hhayi uMshini impela one-ABI ecacile)
isilingo:iwukuhlola okujwayelekile kwe-qPCR.
Idatha ye-qPCR:I-LinRegPCR ingabona izinhlobo ezimbili zamafayela okukhiphayo: i-RDML noma umphumela wokukhulisa ubuningi.Eqinisweni, yinani lesikhathi sangempela lokutholwa kwenombolo yomjikelezo nesiginali ye-fluorescence ngomshini, futhi ukukhulisa kutholwa ngokuhlaziya inani lokushintsha kwe-fluorescence yokusebenza kahle kwengxenye yomugqa.
Ukukhetha idatha: Ngokombono, inani le-RDML kufanele lisetshenziswe.Kulinganiselwa ukuthi inkinga yekhompyutha yami ukuthi isofthiwe ayikwazi ukubona i-RDML, ngakho-ke nginenani lokuphumayo le-excel njengedatha yoqobo.Kunconywa ukwenza ukuhlolwa okungekuhle kwedatha kuqala, njengokwehluleka kokwengeza amasampula, njll. Amaphuzu angasuswa kudatha yokukhipha (Yebo, awukwazi ukuwasusa, i-LinRegPCR izowaziba lawa maphuzu esigabeni sakamuva)

Ukuthunyelwa kwedatha ye-Fig5 qPCR

Ukukhethwa kwe-Fig6 kwamasampula ekhandidethi

Okokufaka kwedatha:Vula imiphumela yokukhulisa ukufaneleka.xls, → vula i-LinRegPCR → ifayela → funda ku-excel → khetha amapharamitha njengoba kukhonjisiwe kuMfanekiso 7 → KULUNGILE → chofoza thola izisekelo

Izinyathelo ze-Fig7 zokufakwayo kwedatha ye-linRegPCR

Umphumela:Uma kungekho ukuphindaphinda, akukho qembu elidingekayo.Uma kukhona ukuphindaphinda, ukuqoqwa kungahlelwa eqenjini lesampula, futhi igama lofuzo lifakwe kusihlonzi, bese kuthi isakhi sofuzo esifanayo siqoqwe ngokuzenzakalelayo.Ekugcineni, chofoza ifayela, thekelisa i-excel, bese ubuka imiphumela.Ukusebenza kahle kokukhulisa kanye nemiphumela engu-R2 yomthombo ngamunye kuzovezwa.Okwesibili, uma uhlukanisa ngamaqembu, ukusebenza kahle kwe-amplification okumaphakathi okulungisiwe kuzoboniswa.Qinisekisa ukuthi ukusebenza kahle kokukhulisa i-primer ngayinye kuphakathi kuka-85% no-115%.Uma inkulu kakhulu noma incane kakhulu, kusho ukuthi ukusebenza kahle kokukhulisa i-primer kubi.

Fig 8 Umphumela kanye nedatha ephumayo

Inqubo yokuhlola:
Izidingo zekhwalithi ye-RNA:
Ubumsulwa:1.7I-2.0 ibonisa ukuthi kungase kube khona i-isothiocyanate esele.I-nucleic acid A260/A230 ehlanzekile kufanele ibe cishe ku-2 .Uma kukhona ukumuncwa okuqinile ku-230 nm, kubonisa ukuthi kunezinhlanganisela eziphilayo ezifana ne-phenate ions.Ngaphezu kwalokho, ingatholwa ngo-1.5% we-agarose gel electrophoresis.Khomba umaka, ngoba i-ssRNA ayinakho ukuchazwa kwe-denaturation futhi i-logarithm yesisindo se-molecular ayinabo ubudlelwano bomugqa, futhi isisindo se-molecular asikwazi ukuvezwa ngendlela efanele.Ukugxila: Ngokwethiyorihhayingaphansi kuka-100ng/ul, uma ukugxilisa ingqondo kuphansi kakhulu, ubumsulwa ngokuvamile buphansi hhayi ubude

Ijeli ye-Fig9 RNA

Ngaphezu kwalokho, uma isampula liyigugu futhi ukugxiliswa kwe-RNA kuphezulu, kuyanconywa ukuthi uliquote ngemva kokukhipha, futhi uhlambulule i-RNA ekugxiliseni kokugcina okungu-100-300ng/ul ukuze ubhale ngokuhlehla.Kuinqubo yokuhlehla okulotshiweyo, lapho kubhalwa i-mRNA, ama-primers we-oligo (dt) angabophezela ngokuqondile kumsila we-polyA asetshenziselwa ukuloba okuhlanekezelwe, kuyilapho i-lncRNA ne-circRNA zisebenzisa ama-primers we-hexamer (Random 6 mer) ekulotshweni okuhlanekezelwe kwengqikithi ye-RNA Kwe-miRNA, imibhalo ebhalwe nge-miRNA ethize eyi-prime isetshenziselwa i-reverse neck-loop.Izinkampani eziningi manje sezethule izinto eziyisipesheli zokusila.Ngendlela ye-stem-loop, indlela yomsila ilula kakhulu, i-high-throughput, futhi yonga i-reagent, kodwa Umphumela wokuhlukanisa ama-miRNA omndeni ofanayo akufanele ube muhle njengendlela ye-stem-loop.Ikhithi ngayinye yokubhala ngokuhlehlayo inezimfuneko zokugxilisa ama-primer aqondene nofuzo (stem-loops).Ireferensi yangaphakathi esetshenziselwa i-miRNA yi-U6.Enqubweni ye-stem-loop inversion, ishubhu le-U6 kufanele lihlehliswe ngokuhlukana, futhi ama-primers angaphambili nangemuva e-U6 kufanele afakwe ngokuqondile.Kokubili i-circRNA ne-lncRNA ingasebenzisa ama-HKG njengereferensi yangaphakathi.Kuukutholwa kwe-cDNA,
uma ingekho inkinga nge-RNA, i-cDNA kufanele futhi ilunge.Kodwa-ke, uma ukuphelela kokuhlolwa kulandelwa, kungcono kakhulu ukusebenzisa isakhi sofuzo sangaphakathi esiyisethenjwa (Reference gene, RG) esingahlukanisa i-gDNA kuma-cd.Ngokuvamile, i-RG iwufuzo lokugcina indlu., HKG) njengoba kukhonjisiwe kuMfanekiso 10;Ngaleso sikhathi, ngangenza amaprotheni okugcina isoya, futhi ngasebenzisa i-actin7 equkethe ama-intron njengereferensi yangaphakathi.Usayizi wocezu olukhulisiwe lwale primer ku-gDNA bekungu-452bp, futhi uma i-cDNA isetshenziswe njengesifanekiso, bekungu-142bp.Khona-ke imiphumela yokuhlola yathola ukuthi Ingxenye ye-cDNA empeleni yayingcoliswe i-gDNA, futhi yafakazela ukuthi kwakungekho nkinga ngomphumela wokuhlehla kokuloba, futhi ingasetshenziswa njengesifanekiso se-PCR.Akusizi ukusebenzisa i-agarose gel electrophoresis ngqo ne-cDNA, futhi iyibhande elisakazwayo, elingagculisi.

Ukutholwa kwe-cDNA Fig 10

Ukunqunywa kwezimo ze-qPCRngokuvamile akuyona inkinga ngokuya ngephrothokholi yekhithi, ikakhulukazi esinyathelweni senani le-tm.Uma amanye ama-primers aklanyelwe kahle ngesikhathi sokuklanywa kwe-primer, okuholela kumehluko omkhulu phakathi kwevelu ye-tm kanye nethiyori engu-60 ° C, kunconywa ukuthi i-cDNA Ngemva kokuba amasampula exutshwe, sebenzisa i-gradient PCR ngama-primer, futhi uzame ukugwema ukusetha izinga lokushisa ngaphandle kwamabhande njengenani le-TM.

Ukuhlaziywa kwedatha

Indlela evamile yokucubungula i-PCR yesihlobo se-fluorescence quantitative ihambisana noku-2-ΔΔCT.Isifanekiso sokucubungula idatha.

Imikhiqizo Ehlobene:

Isikhathi Sangempela PCR Easy^TM – Taqman

Isikhathi Sangempela PCR Easy^TM -SYBR GREEN I

I-RT Easy I (I-Master Premix yokuqala ye-cDNA synthesis)

I-RT Easy II(I-Master Premix yokuqala ye-cDNA synthesis ye-qPCR)