Uvavanyo lwe-RT-qPCR lubandakanya ukutsalwa kwe-RNA kunye novavanyo lomgangatho, ukukhutshelwa umva kunye ne-qPCR amanyathelo amathathu, inyathelo ngalinye linezilumkiso ezininzi, siya kwazisa ngokweenkcukacha ngezantsi.

Ⅰ.Uvavanyo lomgangatho we-RNA

Kuvavanyo lwe-RT-qPCR, emva kokugqitywa kokutsalwa kwe-RNA, umgangatho we-RNA kufuneka uvavanywe, kwaye uvavanyo lokulandelela lunokwenziwa kuphela emva kokuba lufanelekile.Iindlela zokuvavanya ziquka i-spectrophotometer, i-Agilent gel electrophoresis, uhlalutyo lwe-Agilent 2100, phakathi kweyona nto isetyenziswa ngokuqhelekileyo i-spectrophotometer kunye ne-agarose gel electrophoresis indlela yokubona.Kufuneka kuqatshelwe ukuba ezi ndlela zimbini kufuneka zisetyenziswe kunye ukugqiba ukufunyanwa kunye nohlalutyo lwe-RNA yoxinaniso, ukucoceka kunye nokunyaniseka, ukwenzela ukuba kuqinisekiswe umgangatho we-RNA.

Ikhithi yokuKwahlula ye-RNA enxulumeneyo: 

ISeli iyonke yeRNA Isolation Kit

Ukuhlanjululwa okuphezulu kunye nomgangatho ophezulu we-RNA iyonke inokufumaneka kwiiseli ezahlukeneyo zenkcubeko kwi-11min.

Animal Total RNA Isolation Kit

Ngokukhawuleza nangempumelelo ukukhupha ubunyulu obuphezulu kunye nomgangatho ophezulu we-RNA kwizicubu ezahlukeneyo zezilwanyana.

I-Spectrophotometer:

I-spectrophotometer isetyenziselwa ubukhulu becala ukumisela ukugxila kunye nokucoceka kwe-RNA, kodwa ayikwazi ukubona ukunyaniseka kwe-RNA kunye ne-genomic residu.Phakathi kwazo, i-A260/280 kunye ne-A260/230 ziiparamitha ezibalulekileyo zokubona ubunyulu be-RNA, kwaye ubunyulu be-RNA bunokubonwa ngokuguquguquka kwamaxabiso abo:

1. 1.9< A260/280< 2.1, ebonisa ukuba ukucoceka kwe-RNA kulungile;I-A260/280<1.9, ebonisa ukuba kunokubakho i-protein eseleyo kwi-RNA;I-A260/280>2.1, ebonisa ukuthotywa kwe-RNA enokwenzeka, enokuthi iqinisekiswe ngakumbi nge-agarose gel electrophoresis.

2. 2.0< A260/230< 2.2, ebonisa ukuba ukucoceka kwe-RNA kulungile;I-A260/230< 2.0, ebonisa ukuba kunokubakho iintsalela ze-organic reagents kwi-RNA, njenge-phenols, i-ethanol okanye ishukela.

I-Agarose gel electrophoresis:

I-Agarose gel electrophoresis assay inokuhlalutya ingqibelelo ye-RNA, i-genome kunye ne-protein residues, kodwa ayikwazi ukulinganisa ngokuchanekileyo i-concentration ye-RNA okanye ibone i-reagents yezinto eziphilayo.Thatha itemplates ze-eukaryotic RNA umzekelo:

1. I-RNA yayiphantsi kwe-agarose gel electrophoresis.Ukuba bekukho iibhendi ezintathu kuphela ze-28sRNA, 18sRNA kunye ne-5.8sRNA kwimephu yejeli, ibonisa ukuba i-RNA ekhutshiweyo ilungile.Ukuba kukho isenzeko sokutsala, oko kubonisa ukuthotywa kwe-RNA.

2. Ukuba kukho ibhanti enye eqaqambileyo phakathi komngxuma weglue kunye nebhendi ye-28sRNA, kunokubakho intsalela ye-DNA ye-genomic.

3. Ukuba iibhendi zivela kwi-glue hole, kubonisa ukuba kunokubakho iintsalela zeprotheni kunye nezinye izinto ze-macromolecular.

Ⅱ. Reverse transcription

Emva kokuba utsalo lwe-RNA lugqityiwe, kufuneka lutshintshwe lube yi-cDNA kwiimvavanyo ezilandelayo, ngoko ke inyathelo lokubuyisela umva libalulekile.I-reverse transcript iya kwaziswa ekukhethweni kwe-reverse transcriptase kunye ne-primer:

Ukubuyisela umva ukhetho lokukhutshelwa:

Imibhalo eqhelekileyo ebuyela umva ibandakanya i-AMV RTase kunye ne-MMLV RTase.I-RNase H ye-AMV RTase inomsebenzi oqinileyo, ubude obufutshane bokudibanisa, inani eliphantsi lokudibanisa kunye nokuzinza okulungileyo kwe-thermal (42 ~ 55 ℃).Umsebenzi we-RNase H we-MMLV RTase ubuthathaka, ubude be-synthesis bude, inani le-synthesis liphezulu, kwaye ukuzinza kwe-thermal kubi (37 ~ 42 ℃).

Ngenxa yokuba i-enzyme ye-RNase H inomsebenzi wokunciphisa i-template ye-RNA, i-MMLV enobuthakathaka be-RNase H umsebenzi kufuneka ikhethwe ngokukhethekileyo ngexesha lokubhalwa kwe-reverse, kwaye emva kobunjineli bezofuzo kamva, ukuzinza kwe-thermal ye-MMLV kuye kwafikelela kwinqanaba elisemgangathweni.Ukuthatha iForegeneI-Foreasy Reverse Transcriptase(M-MLV yokukhuphela umva) njengomzekelo, yi-reverse transcriptase entsha echazwe kwi-E. coli iibhaktheriya ezenziwe ngobunjineli zisebenzisa iteknoloji yokubuyisela kwakhona ufuzo.Yi-polymerase ye-DNA edibeneyo eyenza i-DNA strand ehambelanayo ukusuka kwi-RNA enye, i-DNA, okanye i-RNA: i-DNA hybrid.Ayinamsebenzi we-RNase H, uzinzo olomeleleyo, ubudlelwane obuqinileyo be-RNA, kunye nokuqonda okuphezulu.

 

I-Foreasy Reverse Transcriptase(M-MLV yokukhuphela umva)

Ukukhethwa kweprimer:

Ngokubanzi iiprimers zeRT ziwela kwiindidi ezintathu: i-oligo dT, iiprimers ezingahleliweyo, kunye ne-gene-specific primers.Khetha iiprimer ezifanelekileyo zokusetyenziswa ngokweemfuno ezahlukeneyo zovavanyo.

1. Ukuba ithempleyithi inemvelaphi ye-eukaryotic kwaye i-cDNA kade isetyenziselwa ukukhulisa i-PCR yesiqhelo, i-Oligo (dT) iyacetyiswa;Ukuba uvavanyo olulandelayo lusetyenziselwa kuphela i-qPCR, i-Oligo (dT) iyacetyiswa ukuba ixutywe ne-random primers ukuphucula ukusebenza kakuhle kokukhutshelwa umva.

2. Ukuba ithempleyithi ivela kwiprokaryotes, ii-Random Primers okanye ii-gene specific primers kufuneka zikhethwe ukwenzela ukubhalwa kwe-reverse.

Ⅲ.qPCR

Ubalo lwe-Fluorescence lucaciswa ikakhulu ekukhethweni kweendlela zobungakanani, imigaqo yoyilo lweprimer, ukhetho lwe-ROX, ukucwangciswa kwenkqubo yokusabela kunye nokusetwa kweemeko zokusabela, njl.

Ukukhethwa kweendlela zobungakanani:

Iindlela zobungakanani bohlulwe ngokweendlela zobungakanani obunxulumeneyo kunye neendlela zobungakanani obupheleleyo.I-quantification ehambelanayo ingasetyenziselwa ukufumanisa umphumo weendlela ezithile zonyango kwi-gene expression, ukubona umehluko we-gene expression kumaxesha ahlukeneyo kwaye uqhathanise umehluko we-gene expression kwii-tissue ezahlukeneyo.Ubungakanani obupheleleyo bunokubona inani le-nucleic acid kwintsholongwane kunye nokunye.Xa sisenza imifuniselo, kufuneka sikhethe iindlela ezifanelekileyo zobungakanani ngokweemvavanyo zethu.

Imigaqo yoyilo yokuqala:

Uyilo lwe-primer ye-qPCR lunxulumene ngokuthe ngqo nokuphumelela kokukhulisa kunye neenkcukacha zemveliso.Ke ngoko, ukuyila ngokuchanekileyo iiprimer ezilungileyo linyathelo lokuqala le-qPCR eyimpumelelo.Kuyilo lwe-primer, le migaqo ilandelayo kufuneka ithathelwe ingqalelo xa idibana nomgaqo woyilo oluqhelekileyo lwe-primer:

1. Ubude beqhekeza elijoliswe kuyo lilawulwa phakathi kwe-100 kunye ne-300 bp;

2. Uyilo lwe-cross-exon ukuphepha impembelelo ye-genomic DNA;

3. Iiprimers eziyilelweyo kufuneka zivavanyelwe ukusebenza kakuhle kwe-amplification, kwaye kuphela xa i-amplification ye-amplification ifikelela kumgangatho (90-110%) ingasetyenziselwa ii-quantitative experiments;

4. I-Primer concentration idla ngokulungiswa phakathi kwe-0.1uM kunye ne-1.0uM.

Ukukhethwa kweI-ROX:

Kwinkqubo yokusabela kobungakanani, i-ROX inokulungelelanisa umahluko wendlela ye-optical, impazamo yombhobho okanye umahluko wevolumu obangelwa kukuphuma komphunga kunye ne-condensation ngokufanayo, ukuphucula ukuphindaphinda kweziphumo.Nangona kunjalo, kufuneka kuqatshelwe ukuba ukhetho lwe-ROX lunxulumene nesixhobo.Ukuba isixhobo se-qPCR sinomsebenzi wokulungisa ngokuzenzekelayo umahluko phakathi kwemingxuma, akufuneki ukuba yongeze i-ROX;kungenjalo, kufuneka yongeze ROX ulungiso.Amaqabane amancinci ekuthengeni ii-reagents kufuneka ahambelane nesixhobo esisetyenziselwa ukukhetha i-ROX echanekileyo, ziphephe iimpazamo zamva.

Ukulungiswa kwenkqubo yokusabela:

Umthamo wokuphendula we-20ul kunye ne-50ul ukhethwayo.Le miba ilandelayo kufuneka inikwe ingqwalasela xa isistim isenziwa:

1. Inkqubo yokusabela kufuneka ilungiswe ngokungena komoya kwindawo yokusebenza ecocekileyo, i-ddH entsha.₂O isetyenziswa kumfuniselo ngamnye;

2. Umfuniselo ngamnye kufuneka ulungiselele i-NTC ukuqinisekisa ukuba ngaba kukho ungcoliseko kwisistim, kwaye iperi nganye yeeprimers kufuneka yenze i-NTC xa ilungiselela isistim;

3. Ukubona ukuba kukho intsalela ye-gDNA kwi-template ye-RNA, i-NRT inokulungiswa kwisampuli nganye ukuze ibonwe;

4. Xa ulungiselela inkqubo, kucetyiswa ukuba wenze ubuncinane i-3 ukuphindaphinda kwezobugcisa kwisampuli enye;

5. Xa ithempleyithi iyi-cDNA, kucetyiswa ukuba kuhlanjululwe amaxesha angama-5-10 ukunciphisa ifuthe lokuthintela lenkqubo yokukhuphela umva kuvavanyo lwe-qPCR.Kungcono ukuhlolisisa ubuninzi betemplate nge-gradient, ukwenzela ukuba ixabiso le-CT liwela phakathi kwe-20-30;

6. Ukugqiba inani elifunekayo lokuphendula, ukunyuka kwe-5-10% kwisiseko senani lokuphendula, kwaye ubale inombolo yokumisela umthamo;

I-7, inkqubo ilungiselelwe ngokusebenzisa umgaqo we-premix, ukuxuba emva kwe-centrifugation kunye nokuqinisekisa ukuba akukho bhubhu;

8, kangangoko kunokwenzeka ukukhetha izinto ezisetyenziswayo ezixhasayo.

Eyeleleneyo RT-qPCR Kit