一、 Yandisa uvakalelo lwenkqubo yokusabela:

1. Yahlula i-RNA ekumgangatho ophezulu:

Impumelelo ye-cDNA synthesis ivela kwi-RNA ekumgangatho ophezulu.Umgangatho ophezulu we-RNA kufuneka ubuncinci bube bubude obugcweleyo kwaye ingabinazo ii-reverse transcriptase inhibitors ezifana ne-EDTA okanye i-SDS.Umgangatho we-RNA umisela ubuninzi beenkcukacha zolandelelwano onokuthi ulukhuphele kwi-cDNA.Indlela yokucoca i-RNA eqhelekileyo yindlela yesinyathelo esisodwa usebenzisa i-guanidine isothiocyanate / i-acid phenol.Ukuthintela ungcoliseko ngamanani omkhondo we-RNase, i-RNA eyodwa kwiisampuli ezityebileyo ze-RNase (ezifana ne-pancreas) kufuneka zigcinwe kwi-formaldehyde ukuze kugcinwe i-RNA ephezulu, ngakumbi ukugcinwa kwexesha elide.I-RNA ekhutshwe kwisibindi segundane yachithwa ngokusisiseko emva kokugcinwa emanzini iveki enye, ngelixa i-RNA ekhutshwe kwi-rat spleen yahlala izinzile emva kokugcinwa emanzini iminyaka eyi-3.Ukongeza, imibhalo emide kune-4 kb inovakalelo ngakumbi ekuthotyweni ngokulandela i-RNases kunemibhalo emincinci.Ukwandisa ukuzinza kweesampuli ze-RNA ezigciniweyo, i-RNA inokuchithwa kwi-formamide ye-deionized kwaye igcinwe kwi -70 ° C.IFormamide esetyenziselwa ukulondoloza i-RNA kufuneka ingabi nankunkuma ye-RNA ethoba isidima.I-RNA esuka kwi-pancreas inokugcinwa kwi-formamide ubuncinane unyaka omnye.Xa ulungiselela ukusebenzisa i-RNA, ungasebenzisa le ndlela ilandelayo yokunyusa i-RNA: yongeza i-NaCl kwi-0.2M kunye namaxesha ama-4 umthamo we-ethanol, ubeke kwindawo yokushisa kwe-3-5 imizuzu, kunye ne-centrifuge kwi-10,000 × g imizuzu emi-5.

2. Sebenzisa i-RNaseH-inactive (RNaseH-) reverse transcriptase:

I-RNase inhibitors zihlala zongezwa ukubuyisela umva ukuphendula okushicilelweyo ukwandisa ubude kunye nemveliso ye-cDNA synthesis.I-RNase inhibitors kufuneka yongezwe ngexesha le-first-strand synthesis reaction phambi kwe-buffer kunye ne-arhente yokunciphisa (efana ne-DTT), kuba inkqubo ngaphambi kwe-cDNA synthesis denatures inhibitor, ngaloo ndlela ikhulula i-RNase eboshiweyo enokuthi ithobe i-RNA.Iprotheyini ye-RNase inhibitors ikhusela kuphela ukuthotywa kwe-RNA nge-RNase A, B, C, kwaye ayithinteli i-RNase elukhumbeni, ngoko qaphela ukuba ungafaki i-RNase kwiminwe yakho nangona ukusetyenziswa kwezi inhibitors.

I-reverse transcriptase yenza ukuguqulwa kwe-RNA ibe yi-cDNA.Zombini i-M-MLV kunye ne-AMV zinomsebenzi we-RNaseH ongapheliyo ukongeza kumsebenzi wazo wepolymerase.Umsebenzi we-RNaseH kunye nomsebenzi we-polymerase ukhuphisana omnye nomnye kwi-hybrid strand eyenziwe phakathi kwe-template ye-RNA kunye ne-DNA primer okanye i-cDNA extension strand, kunye nokunciphisa i-RNA strand kwi-RNA: DNA complex.Ithemplate ye-RNA ethotywe ngumsebenzi we-RNaseH ayinakuphinda isebenze njenge-substrate esebenzayo ye-cDNA synthesis, enciphisa isivuno kunye nobude be-cDNA synthesis.Ke ngoko, kuya kuba luncedo ukuphelisa okanye ukunciphisa kakhulu umsebenzi we-RNaseH we-reverse transcriptase..

I-SuperScript Ⅱ i-reverse transcriptase, i-RNaseH- MMLV i-reverse transcriptase kunye ne-thermoScript reverse transcriptase, i-RNaseH- AMV, inokufumana imali eninzi kunye ne-cDNA epheleleyo kune-MMLV kunye ne-AMV.Uvakalelo lwe-RT-PCR luya kuchaphazeleka ngubungakanani be-cDNA synthesis.I-ThermoScript ibuthathaka kakhulu kune-AMV.Ubungakanani beemveliso ze-RT-PCR bukhawulelwe kukukwazi kwe-reverse transcriptase ukudibanisa i-cDNA, ngakumbi xa udibanisa ii-cDNAs ezinkulu.Xa kuthelekiswa neMMLV, iSuperScripⅡ yandise kakhulu isivuno seemveliso ezinde zeRT-PCR.I-RNaseH-reverse transcriptase nayo inyuse izinga lobushushu, ngoko ke ukusabela kunokwenziwa kumaqondo obushushu angaphezulu kwama-37-42°C.Ngaphantsi kweemeko eziphakanyisiweyo zokwenziwa, sebenzisa i-oligo (dT) primer kunye ne-10 μCi ye- [α-P] dCTP.Isivuno esipheleleyo se-strand yokuqala sibalwa kusetyenziswa indlela ye-TCA yemvula.I-cDNA yobude obugcweleyo yahlalutywa kusetyenziswa iibhanti ezihlelwe ngokwesayizi ezikhutshiweyo kwaye zibalwe kwijeli ye-alkaline ye-agarose.

3. Phakamisa ubushushu bokufukamela ukuguqulelwa umva:

Ukushisa okuphezulu kwe-incubation kunceda ukuvula isakhiwo sesibini se-RNA, ukwandisa isivuno sokusabela.Kwii-templates ezininzi ze-RNA, ukufukamela i-RNA kunye ne-primers kwi-65 ° C ngaphandle kwe-buffer okanye ityuwa, elandelwa ukupholisa ngokukhawuleza kwiqhwa kuya kuphelisa ezininzi izakhiwo zesibini kwaye zivumele ii-primers ukuba zibophe.Nangona kunjalo, ezinye iitemplates zisenezakhiwo zesibini, nasemva kokutshatyalaliswa kobushushu.Ukwandiswa kwezi templates ezinzima kunokwenziwa kusetyenziswa i-ThermoScript Reverse Transcriptase kunye nokubeka impendulo yokukhuphela umva kwiqondo lobushushu eliphezulu ukuphucula ukukhulisa.Amaqondo obushushu aphezulu okufukamela nawo anokonyusa ukucaciswa, ngakumbi xa iiprimers zegene-specific (GSP) zisetyenziselwa ukuhlanganiswa kwe-cDNA (jonga iSahluko 3).Ukuba usebenzisa i-GSP, qinisekisa ukuba i-Tm yeeprimers iyafana nobushushu obulindelekileyo bokufukamela.Musa ukusebenzisa i-oligo(dT) kunye ne-random primers ngaphezu kwe-60°C.Iiprimers ezingaqhelekanga zifuna ukufukanyelwa kwi-25°C kangangemizuzu eyi-10 phambi kokuba zinyuke ziye kuma-60°C.Ukongeza ekusebenziseni iqondo lobushushu obuphezulu lokukhutshelwa kwereverse, ukuchaneka kungaphuculwa ngokugqithisela ngokuthe ngqo umxube we-RNA/primer ukusuka kubushushu obungama-65°C denaturation ukuya kwiqondo lobushushu lereverse loshicilelo lokufukamela kunye nokongeza umxube ofudunyeziweyo we-2× reaction (cDNA hot-start synthesis) .Le ndlela inceda ukukhusela i-intermolecular base pairing eyenzeka kumaqondo okushisa aphantsi.Ukutshintsha kweqondo lokushisa okuninzi okufunekayo kwi-RT-PCR kunokwenziwa lula ngokusebenzisa i-thermal cycler.

I-Tth thermostable polymerase isebenza njengepolymerase ye-DNA phambi kwe-Mg2 + kunye ne-RNA polymerase phambi kwe-Mn2 +.Ingagcinwa ishushu kubushushu obuphezulu obungama 65°C.Nangona kunjalo, ubukho be-Mn2 + ngexesha le-PCR kunciphisa ukuthembeka, okwenza i-Tth i-polymerase ingafaneleki kakhulu kwi-high-precision amplification, njenge-cloning ye-cDNA.Ukongezelela, i-Tth ine-low reverse transcription esebenzayo, enciphisa uvakalelo, kwaye, ekubeni ukuguqulelwa kwe-reverse kunye ne-PCR kunokwenziwa nge-enzyme enye, ukusabela kokulawula ngaphandle kokubhalwa kwe-reverse akunakusetyenziselwa ukuthelekisa iimveliso ze-cDNA zokukhulisa kunye ne-DNA engcolileyo ye-genomic.Iimveliso zokukhulisa zahlulwa.

4. Izongezo ezikhuthaza ukukhutshelwa umva:

Izongezo ezibandakanya i-glycerol kunye ne-DMSO zongezwa kwi-first-strand synthesis reaction, enokunciphisa ukuzinza kwe-nucleic acid double-strand kwaye ikhulule isakhiwo sesibini se-RNA.Ukuya kwi-20% ye-glycerol okanye i-10% ye-DMSO inokongezwa ngaphandle kokuchaphazela umsebenzi we-SuperScript II okanye we-MMLV.I-AMV inokunyamezela ukuya kwi-20% ye-glycerol ngaphandle kokulahlekelwa ngumsebenzi.Ukuze kwandiswe ubuntununtunu be-RT-PCR kwi-SuperScriptⅡ reverse transcription reaction, i-10% ye-glycerol inokudityaniswa kwaye incubated kwi-45°C.Ukuba i-1/10 ye-reverse transcription reaction reaction yongezwa kwi-PCR, ngoko ukuxinwa kwe-glycerol kwi-amplification reaction yi-0.4%, enganelanga ukunqanda i-PCR.

5. RNaseH unyango:

Unyango lwe-cDNA synthesis reactions kunye ne-RNaseH phambi kwe-PCR inokunyusa ubuntununtunu.Kwezinye iitemplates, kucingelwa ukuba i-RNA kwi-cDNA synthesis reaction ithintela ukubophelela kweemveliso zokukhulisa, apho unyango lwe-RNaseH lunokwandisa ubuntununtunu.Ngokubanzi, unyango lwe-RNaseH luyimfuneko xa kusandiswa itemplates ezijolise kubude obugcweleyo be-cDNA, ezifana nekopi ephantsi ye-tuberous scherose II.Kule template inzima, unyango lwe-RNaseH luphucule umqondiso oveliswe yi-SuperScript II okanye i-AMV-synthesized cDNA.Kwiimpendulo ezininzi ze-RT-PCR, unyango lwe-RNaseH luyinketho, kuba inyathelo le-PCR denaturation kwi-95 ° C ngokubanzi i-hydrolyzes i-RNA kwi-RNA: i-DNA complex.

6. Ukuphuculwa kweNdlela yokuFumana iRNA encinci:

I-RT-PCR icela umngeni ngakumbi xa kukho iimali ezincinci ze-RNA ezifumanekayo.I-Glycogen eyongeziweyo njengomthwali ngexesha lokuhlukaniswa kwe-RNA inceda ukwandisa isivuno seesampuli ezincinci.I-glycogen ye-RNase-free inokongezwa ngexesha elifanayo njengokongeza i-Trizol.I-Glycogen iyanyibilika emanzini kwaye inokugcinwa kwinqanaba le-aqueous kunye ne-RNA ukunceda imvula elandelayo.Kwiisampuli ezingaphantsi kwe-50 mg yezicubu okanye iiseli ze-106 ezikhuliswe, i-concentration ekhuthazwayo ye-RNase-free glycogen yi-250 μg / ml.

Ukongeza i-acetylated BSA kwi-reverse transcription reaction usebenzisa i-SuperScript II inokunyusa uvakalelo, kunye nexabiso elincinci le-RNA, ukunciphisa inani le-SuperScript II kunye nokongeza iiyunithi ze-40 ze-RNaseOut nuclease inhibitor inokunyusa izinga lokubhaqa.Ukuba i-glycogen isetyenziswe kwinkqubo yokuhlukaniswa kwe-RNA, kusacetyiswa ukuba yongezwe i-BSA okanye i-RNase inhibitor xa usebenzisa i-SuperScript II ukwenzela ukusabela okushicilelweyo.

二、 Yandisa i-RT-PCR ethile

1. CND Asynthesis:

Ukuhlanganiswa kwe-cDNA yokuqala-strand kunokuqaliswa ngokusebenzisa iindlela ezintathu ezahlukeneyo, ukuchaneka okuhambelanayo okuchaphazela isixa kunye nohlobo lwe-cDNA edibeneyo.

Indlela ye-random primer yayiyeyona incinci kakhulu kwiindlela ezintathu.Iiprimer anneal kwiindawo ezininzi kulo lonke ushicilelo, zenza ii-cDNAs ezimfutshane, ezinobude obungaphelelanga.Le ndlela isetyenziswa rhoqo ukufumana i-5 'yokulandelelana kokuphela kunye nokufumana i-cDNA kwii-templates ze-RNA kunye nemimandla yesakhiwo sesibini okanye kunye neziza zokuphelisa ezingenakuphinda ziphindwe nge-reverse transcriptase.Ukufumana eyona cDNA inde, umlinganiselo we-primers ukuya kwi-RNA kwisampulu nganye ye-RNA kufuneka uqingqwe ngokwamandla.Isiqalo soxinaniso lweeprimers olungenamkhethe lwalusuka kwi-50 ukuya kwi-250 ng nge-20 μl yokusabela.Ekubeni i-cDNA idityaniswe kwi-RNA iyonke kusetyenziswa i-random primers ngokuyintloko yi-ribosomal RNA, i-poly(A)+RNA ikhethwa ngokubanzi njenge template.

Iiprimers ze-Oligo (dT) zithe ngqo ngakumbi kunezokuqala ezingahleliweyo.Idibanisa kwi-poly(A) umsila ofunyenwe kwi-3′ ekupheleni kwezona zininzi ze-eukaryotic mRNAs.Ngenxa yokuba i-poly(A)+ i-RNA imalunga ne-1% ukuya kwi-2% ye-RNA iyonke, isixa kunye nobunzima be-cDNA bungaphantsi kakhulu kune-random primers.Ngenxa yokucaciswa kwayo okuphezulu, i-oligo(dT) ayifuni ngokubanzi ukulungelelaniswa komlinganiselo we-RNA kwii-primers kunye ne-poly(A)+ yokukhetha.Kucetyiswa ukuba usebenzise i-0.5μg oligo (dT) nge-20μl inkqubo yokusabela.i-oligo(dT)12-18 ilungele uninzi lwe-RT-PCR.I-ThermoScript RT-PCR System ibonelela nge-oligo(dT)20 ngenxa yozinzo olungcono lwe-thermal kumaqondo aphezulu obushushu bokufukanyelwa.

Ii-primers ze-Gene specific (GSP) zezona zi-primers ezithe ngqo kwinyathelo lokukhuphela umva.I-GSP yi-oligonucleotide ye-antisense enokuthi idibanise ngokuthe ngqo ukulandelelana kwethagethi ye-RNA, ngokungafaniyo ne-random primers okanye i-oligo (dT), edibanisa zonke ii-RNAs.Imithetho efanayo esetyenziselwa ukuyila iiprimer zePCR iyasebenza kuyilo lwe-GSP kwireverse transcription reactions.I-GSP inokuba nolandelelwano olufanayo njenge-primer yokukhulisa i-anneals ukuya kwi-3'-eyona siphelo se-mRNA, okanye i-GSP inokuyilwa ukuba idibanise ezantsi kwe-primer yokukhulisa umva.Kwizifundo ezithile ezandisiweyo, ngaphezu kwe-primer enye ye-antisense kufuneka yenzelwe i-RT-PCR eyimpumelelo kuba isakhiwo sesibini se-RNA ekujoliswe kuyo sinokuthintela ukubopha i-primer.Kucetyiswa ukuba kusetyenziswe i-1 pmol antisense GSP kwi-20 μl yokuqala yokusabela kwe-strand synthesis.

2. Phakamisa ubushushu bokufukamela ukuguqulelwa umva:

Ukuze kusetyenziswe ngokupheleleyo inzuzo epheleleyo ye-GSP, i-reverse transcriptase ene-thermostability ephezulu kufuneka isetyenziswe.I-Thermostable reverse transcriptases inokufukanyelwa kumaqondo obushushu aphezulu ukonyusa ukuqina kokusabela.Umzekelo, ukuba i-GSP ithatha i-55°C, iinkcukacha ze-GSP aziyi kusetyenziswa ngokupheleleyo ukuba i-AMV okanye i-M-MLV isetyenziselwa ukukhutshelwa umva ngoxinzelelo oluphantsi lwama-37°C.Nangona kunjalo, i-SuperScript II kunye ne-ThermoScript inokuphendulwa kwi-50 ° C okanye ngaphezulu, eya kuphelisa iimveliso ezingezizo ezikhethekileyo ezenziwe kumaqondo aphantsi.Ukuchaneka okuphezulu, umxube we-RNA/primer ungagqithiswa ngokuthe ngqo ukusuka kwi-65 ° C denaturation ubushushu ukuya kwiqondo lobushushu lokukhuphela elibuyisela umva kwaye longezwe kumxube wokusabela oshushu we-2× (ukuqala kwe-cDNA synthesis eshushu).Oku kunceda ukukhusela isiseko se-intermolecular kumaqondo obushushu aphantsi.Iinguqu ezininzi zobushushu ezifunekayo kwi-RT-PCR zingenziwa lula ngokusebenzisa i-thermal cycler.

3. Yehlisa ukosuleleka kwe-DNA ye-genomic:

Ubunzima obunokubakho bokudibana ne-RT-PCR kukungcoliseka kwe-genomic DNA kwi-RNA.Ukusebenzisa indlela efanelekileyo yokuhlukanisa i-RNA, njenge-Trizol Reagent, iya kunciphisa inani le-genomic DNA engcolisa ukulungiswa kwe-RNA.Ukuze ugweme iimveliso ezivela kwi-DNA ye-genomic, i-RNA inokunyangwa nge-amplification-grade DNase I ukususa i-DNA engcolisayo ngaphambi kokuguqulelwa umva.I-DNase I digestion yapheliswa ngokufukamela iisampuli kwi-2.0 mM EDTA ngemizuzu eyi-10 kwi-65 ° C.I-EDTA inokutshisa i-magnesium ion, ukukhusela i-RNA hydrolysis exhomekeke kwi-ion kubushushu obuphezulu.

Ukuze kwahlulwe i-cDNA eyandisiweyo ekungcoliseni iimveliso zokukhulisa i-DNA ye-genomic, iiprimers zinokuyilwa ukuba i-anneal nganye yahlule ii-exons.Iimveliso ze-PCR ezivela kwi-cDNA ziya kuba mfutshane kunezo zivela kwi-DNA ye-genomic engcolileyo.Ukongeza, uvavanyo lokulawula ngaphandle kokukhutshelwa umva lwenziwa kwitemplate nganye ye-RNA ukujonga ukuba iqhekeza elinikiweyo lithathwe kwi-genomic DNA okanye i-cDNA.Imveliso ye-PCR efunyenwe ngaphandle kwe-reverse transcription ivela kwi-genome.