Ⅰ. Yandisa uvakalelo lwenkqubo yokusabela:

1. Yahlula iRNA yomgangatho ophezulu:

Impumelelo ye-cDNA synthesis ivela kwi-RNA ekumgangatho ophezulu.I-RNA yomgangatho ophezulu kufuneka iqinisekise ubuncinci ixesha elide kwaye ayiqulathanga i-inhibitors engenazo ii-enzymes zokurekhoda, ezifana ne-EDTA okanye i-SDS.Umgangatho we-RNA umisela elona xabiso liphezulu lolwazi lolandelelwano onokuthi ulubhale kwi-cDNA.Indlela yokucoca i-RNA ngokubanzi yindlela yenyathelo lokusebenzisa i-isoocyanate/acidophenol.Ukuze kuthintelwe ukungcoliseka kwe-RNase, i-RNA ehlulwe kwisampulu ecebileyo kwi-RNase (efana ne-pancreas) idinga ukugcinwa kwe-formaldehyde ukugcina i-RNA ephezulu, eyona nto ibaluleke kakhulu yokugcina ixesha elide.I-RNA ekhutshwe kwisibindi segundane yachithwa ngokusisiseko emva kweveki enye yokugcina emanzini, ngelixa i-RNA ekhutshwe kwi-rat spleen yahlala izinzile emva kweminyaka emithathu yokugcina emanzini.Ukongeza, imibhalo emikhulu kune-4kb inovakalelo ngakumbi ekulandeleni ukuthotywa kwe-RNase kunemibhalo emincinci.Ukuze ukwandise ukuzinza kwesampulu ye-RNA yokugcina, i-RNA inokuchithwa kwi-methalmamine ye-ion, kwaye igcinwe -70 °C.I-Thylide esetyenzisiweyo ukugcina i-RNA mayingaqulathi izinto ezahlukeneyo ezehlisa isidima se-RNA.I-RNA, ephuma kwi-pancreas, inokugcinwa kwi-methalmamine ubuncinane unyaka omnye.Xa ulungele ukusebenzisa i-RNA, ungasebenzisa ezi ndlela zilandelayo ukuze uqhube i-RNA: yongeza i-NaCl kwi-0.2m kunye namaxesha ama-4 umthamo we-ethanol, indawo yokushisa kwegumbi le-3-5 imizuzu, kunye ne-10,000 × g centrifugal imizuzu emi-5.

2. Sebenzisa i-reverse transcriptase ngaphandle komsebenzi we-RNaseH (RNaseH-):

I-RNase inhibitors zihlala zongezwa ukubuyisela umva ukuphendula okushicilelweyo ukwandisa ubude kunye nemveliso ye-cDNA synthesis.I-RNase inhibitor yongezwa kwi-rection ye-chain synthesis yokuqala phambi kwee-buffers kunye nee-arhente zokunciphisa ezifana ne-DTT kuba inkqubo ye-pre-cDNA synthesis denatures inhibitor, ngaloo ndlela ikhulula i-RNases eboshiweyo ethoba i-RNA.I-protein RNase inhibitor ikhusela kuphela ukuthotywa kwe-RNA nge-RNase A, B, C, kwaye ayithinteli i-RNase esikhumbeni, ngoko ke kufuneka kuthathelwe ingqalelo ukuba ungafaki i-RNases kwiminwe 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 ukhuphisana nomsebenzi we-polymerase kwiintambo ze-heterozygous ezenziwe phakathi kwee-templates ze-RNA kunye ne-DNA primers okanye i-cDNA extension strands, kunye nokunciphisa i-RNA: i-RNA strands kwi-DNA complexes.Iitemplates ze-RNA ezonakaliswe ngumsebenzi we-RNaseH azinakuphinda zisetyenziswe njengama-substrates asebenzayo kwi-cDNA synthesis, ukunciphisa isivuno kunye nobude be-cDNA synthesis.Ngaloo ndlela ukuphelisa okanye ukunciphisa kakhulu umsebenzi we-RNaseH we-reverse transcriptase kuya kuba luncedo olukhulu.

I-SuperScriptⅡ i-reverse transcriptase, i-MMLV reverse transcriptase ye-RNaseH- kunye ne-thermoScript reverse transcriptase, i-AMV ye-RNaseH- ivelise i-cDNA yobude obugcweleyo ngaphezu kwe-MMLV kunye ne-AMV.Uvakalelo lwe-RT-PCR luchatshazelwa ngubungakanani be-cDNA edibeneyo.I-ThermoScript ibuthathaka kakhulu kune-AMV.Ubungakanani beemveliso ze-RT-PCR bukhawulelwe kukukwazi kwe-reverse transcriptase ukudibanisa i-cDNA, ngakumbi xa uhlanganisa ii-Cdna ezinkulu.Xa kuthelekiswa neMMLV, iSuperScripⅡ yandise kakhulu isivuno seemveliso ezinde zeRT-PCR.I-RNaseH-'s reverse transcriptase ikwanyusa uzinzo lwe-thermal, ngoko ke ukusabela kunokwenziwa kumaqondo obushushu aphezulu kunesiqhelo angama-37-42℃.Phantsi kweemeko ezicetyisiweyo zokudityaniswa, iiprimers ze-oligo (dT) kunye ne-10μCi [alpha-p] dCTP zisetyenzisiwe.Imveliso epheleleyo yekhonkco yokuqala ibalwe kusetyenziswa indlela ye-TCA yemvula.I-cDNA yobude obupheleleyo yahlalutywa kusetyenziswa ukususwa komcu ohleliweyo ngokobungakanani nokubalwa kwijeli yealkaline yeagarose.

3. Yongeza ubushushu bokugcina ubushushu bokukhutshelwa umva:

Ukushisa okuphezulu okubambayo kunceda ukuvula isakhiwo sesibini se-RNA kunye nokwandisa isivuno sokuphendula.Kwii-templates ezininzi ze-RNA, ukubamba i-RNA kunye ne-primer kwi-65 ° C ngaphandle kwe-buffer okanye ityuwa kwaye emva koko ukupholisa ngokukhawuleza kwiqhwa kuphelisa ezininzi izakhiwo zesibini kwaye zivumela iinqununu ukuba zibophe.Nangona kunjalo, ezinye iitemplates zisenolwakhiwo lwesibini, nasemva kwe-thermal denaturation.Ukwandiswa kwezi templates ezinzima kunokwenziwa kusetyenziswa i-ThermoScript reverse transcriptase kwaye ngokubeka i-reverse transcriptase reaction kumaqondo obushushu aphezulu ukuphucula ukukhulisa.Amaqondo okushisa aphezulu anokuthi anyuse ngokukodwa, ngakumbi xa i-cDNA synthesis yenziwa kusetyenziswa i-gene-specific primers (GSPS) (jonga iSahluko 3).Ukuba usebenzisa i-GSP, qiniseka ukuba ixabiso le-Tm le-primer liyafana neqondo lokushisa elilindelekileyo.Musa ukusebenzisa i-oligo(dT) kunye ne-random primers ngaphezulu kwe-60℃.Iiprimers ezingaqhelekanga kufuneka zigcinwe kwi-25℃ imizuzu eyi-10 phambi kokuba zinyuke ziye kuma-60℃.Ukongeza ekusebenziseni amaqondo obushushu aphezulu oshicilelo oluguqulelweyo, ukuchaneka kungaphuculwa ngokugqithisela ngokuthe ngqo umxube we-RNA/primer ukusuka kwi-65℃ denaturing iqondo lobushushu ukuya kwiqondo lobushushu eliguqulelweyo kunye nokongeza umxube we-2 × wokusabela kwangaphambili (cDNA thermal initiation synthesis).Le ndlela inceda ukukhusela i-intermolecular base pairing eyenzeka kumaqondo okushisa aphantsi.Ukusebenzisa isixhobo sePCR kwenza lula utshintsho oluninzi lobushushu olufunekayo kwi-RT-PCR.

Ubushushu obuzinzile bepolymerase busebenza njenge-DNA polymerase phambi kweMg2 + kunye neRNA polymerase phambi kweMn2 +.Iyakwazi ukubamba ubushushu ukuya kuma 65℃.Nangona kunjalo, ubukho be-Mn2 + ngexesha le-PCR kunciphisa ukuthembeka, okwenza i-Tth i-polymerase ingafanelanga ukunyusa okuchanekileyo okuphezulu, njenge-cDNA cloning.Ukongezelela, i-Tth ayisebenzi kakuhle kwi-reverse transcription, enciphisa uvakalelo, kwaye ekubeni i-enzyme enye inokwenza i-reverse transcription kunye ne-PCR, iimpendulo zokulawula ngaphandle kwe-reverse transcription ayinakusetyenziswa ukwahlula iimveliso ezikhulisiwe ze-cDNA kwezo ze-DNA engcolileyo ye-genomic.

4. Isihlomelo esikhuthaza ukukhutshelwa umva:

Ukongezwa kwezongezo, kubandakanywa i-glycerin kunye ne-DMSO, kwi-reaction ye-chain synthesis yokuqala inokunciphisa ukuzinza kwe-nucleic acid strand kabini kunye nokukhulula isakhiwo sesibini se-RNA.Ukuya kuthi ga kwi-20% ye-glycerin okanye i-10% ye-DMSO inokongezwa ngaphandle kokuchaphazela umsebenzi we-SuperScriptⅡ okanye i-MMLV.I-AMV inokunyamezela ukuya kwi-20% ye-glycerol ngaphandle kokunciphisa umsebenzi.Ukwandisa ubuntununtunu be-RT-PCR kwi-SuperScriptⅡ reverse transcription reaction, i-10% ye-glycerol inokongezwa kwaye ifakwe kwi-45℃.Ukuba i-1/10 yemveliso ye-retrotranscription-reaction yongezwa kwi-PCR, i-concentration ye-glycerol kwi-amplification reaction yi-0.4%, enganelanga ukuvimbela i-PCR.

5. Ukulungiswa kwe-RNaseH:

Uvakalelo lunokuphuculwa ngokuphatha i-cDNA synthesis reactions kunye ne-RNaseH phambi kwe-PCR.Kwezinye iitemplates, kucingelwa ukuba i-RNA kwi-cDNA synthesis reaction ithintela ukubotshwa kweemveliso ezikhulisiwe, apho unyango lwe-RNaseH lunokunyusa uvakalelo.Ngokubanzi, unyango lwe-RNaseH luyafuneka ekwandiseni itemplate ye-cDNA yobude obugcweleyo, efana ne-tuberous scherosisⅡ enekopi ephantsi.Kule template inzima, i-RNaseH iphucule umqondiso oveliswe yi-cDNA eyenziwe yiSuperScriptⅡ okanye i-AMV.Kwiimpendulo ezininzi ze-RT-PCR, unyango lwe-RNaseH luyinketho kuba inyathelo le-PCR eligqunyiweyo le-95℃ lenza i-hydrolyzes i-RNA esuka kwi-RNA: i-DNA complex.

6. Iindlela eziphuculweyo zokufumana iimali ezincinci zeRNA:

I-RT-PCR icela umngeni ngakumbi xa kukho iimali ezincinci ze-RNA ezifumanekayo.Ukongezwa kwe-glycogen njengomthwali ngexesha lokuhlukana kwe-RNA kunceda ukwandisa isivuno seesampuli ezincinci.I-glycogen engena-RNase inokongezwa ngexesha elifanayo ne-Trizol.I-Glycogen iyanyibilika emanzini kwaye ingahlala kwisigaba samanzi kunye ne-RNA ukuncedisa kwimvula elandelayo.I-concentration ekhuthazwayo ye-RNase-free glycogen yi-250μg / ml kwiisampuli ezingaphantsi kwe-50mg yezicubu okanye iiseli ezikhuliswe nge-106.

Ukongezwa kwe-acetylated BSA ukubuyisela umva ukuphendula okushicilelweyo kusetyenziswa iSuperScriptⅡ kunokunyusa uvakalelo, kunye nezixa ezincinci zeRNA, ukunciphisa inani leSuperScriptⅡ kunye nokongeza iiyunithi ezingama-40 zeRnaseOut nuclease inhibitor kunokuphucula inqanaba lokubona.Ukuba i-glycogen isetyenziswa kulwahlulo lwe-RNA, ukongezwa kwe-BSA okanye i-RNase inhibitors ukubuyisela umva impendulo yoshicilelo kusetyenziswa i-SuperScriptⅡ isacetyiswa.

Ⅱ. Ukwandisa ukucaciswa kwe-RT-PCR

1. CNDA synthesis:

Iindlela ezintathu ezahlukeneyo zingasetyenziselwa ukuqalisa i-strand yokuqala ye-cDNA synthesis, kunye nokucaciswa okuhambelanayo kwendlela nganye kuchaphazela isixa kunye nohlobo lwe-cDNA edibeneyo.

Indlela ye-primer engaqhelekanga yeyona ndlela incinci kwezi zintathu.Iiprimers zifakwe kwiindawo ezininzi kwi-transcript ukuvelisa i-cDNA emfutshane, ubude obuyinxenye.Le ndlela isoloko isetyenziselwa ukufumana i-5′ ulandelelwano lwe-terminal kunye ne-cDNA kwii-templates ze-RNA ezinemimandla yesakhiwo sesibini okanye kunye neziza zokuphelisa i-reverse transcriptase ayikwazi ukuphindaphinda.Ukufumana eyona cDNA inde, umlinganiselo we-primers ukuya kwi-RNA kwisampulu nganye ye-RNA kufuneka uqingqwe ngokwamandla.Uxinzelelo lokuqala lwee-primers olungenamkhethe lusuka kwi-50 ukuya kwi-250ng nge-20μl inkqubo yokusabela.Ngenxa yokuba i-cDNA idityaniswe kwi-RNA iyonke kusetyenziswa ii-primers ezingahleliwe ikakhulu yi-ribosomal RNA, i-poly(A)+RNA ikhethwa ngokubanzi njenge template.

Ukuqaliswa kwe-Oligo (dT) kungqale ngakumbi kunee-primers ezingahleliwe.Idibanisa i-poly(A) umsila ofunyenwe kwi-3′ ekupheleni kwe-mRNA kwiiseli ezininzi ze-eukaryotic.Ngenxa yokuba i-poly(A)+RNA imalunga ne-1% ukuya kwi-2% ye-RNA iyonke, isixa kunye nobunzima be-cDNA bungaphantsi kakhulu kunokuba kusetyenziswe iiprimers ezingahleliwe.Ngenxa yokuchaneka kwayo okuphezulu, i-oligo(dT) ayifuni ngokubanzi ukwenziwa kwe-RNA kumlinganiselo we-primer 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 yokuzinza kwayo okuhle kwe-thermal kwaye ifanelekile kumaqondo obushushu aphezulu.

Ii-primers ze-Gene-specific (GSP) zezona zi-primers ezikhethekileyo zenyathelo lokukhuphela elibuyela umva.I-GSP yi-oligonucleoside ye-antisense enokuthi idibanise ngokuthe ngqo kunye nolandelelwano lwendawo ye-RNA, kunokuba i-anneal yonke i-Rnas njenge-random primers okanye i-oligo (dT).Imigaqo esetyenziswayo ukuyila iiprimer zePCR ikwasebenza kuyilo lwempendulo yereverse transcription reaction GSP.I-GSP inokuba nolandelelwano olufana ne-primer yokukhulisa i-annealed ekupheleni kwe-mRNA3′, okanye i-GSP inokuyilwa ukuba ifakwe ezantsi kumlambo nge-primer yokukhulisa umva.Kwezinye izinto ezandisiweyo, kuyimfuneko ukuyila ngaphezu kwesinye i-antisense primer ye-RT-PCR eyimpumelelo kuba ulwakhiwo lwesibini lwe-RNA ekujoliswe kuyo lunokuthintela i-primer ekubopheni.Kucetyiswa ukuba kusetyenziswe i-1pmol ye-antisense ye-GSP kwinkqubo yokuqala yokusabela ye-chain synthesis ye-20μl.

2. Yongeza ubushushu bokugcina ubushushu bokukhutshelwa umva:

Ukuze kusetyenziswe ngokupheleleyo i-GSP ethile, i-reverse transcriptase enozinzo oluphezulu lwe-thermal kufuneka isetyenziswe.Ubushushu obuzinzileyo be-reverse transcriptase bunokugqunywa kumaqondo obushushu aphezulu ukunyusa ukuqina kokusabela.Umzekelo, ukuba i-GSP ifakwe kwi-55 ° C, ngoko i-speciality ye-GSP ayisetyenziswanga ngokupheleleyo ukuba i-reverse transcription yenziwa kwi-37 °C ngokungqongqo okuphantsi kusetyenziswa i-AMV okanye i-M-MLV.Nangona kunjalo, iSuperScripⅡ kunye neThermoScript inokusabela kwi-50℃ okanye ngaphezulu, esusa iimveliso ezingezizo eziveliswe kumaqondo obushushu asezantsi.Ukuchaneka okuphezulu, umxube we-RNA / primer unokudluliselwa ngokuthe ngqo kwi-65℃ ye-denaturation yeqondo lokushisa ukuya kwi-reverse transcription ephethe ubushushu kunye nokongezwa kwe-preheated 2 x umxube wokuphendula (ukuqaliswa kwe-thermal ye-cDNA synthesis).Oku kunceda ukuthintela ukubhanqa isiseko phakathi kwama-athomu kumaqondo obushushu aphantsi.Ukusebenzisa isixhobo sePCR kwenza lula utshintsho oluninzi olufunekayo kwi-RT-PCR.

3. Nciphisa ungcoliseko lwe-DNA ye-genomic:

Obunye ubunzima obunokubakho nge-RT-PCR kukuba i-RNA yosulela i-genomic DNA.Ukusetyenziswa kweendlela ezingcono zokuhlukana kwe-RNA, njenge-Trizol Reagent, kunciphisa ukungcoliswa kwe-DNA ye-genomic kumalungiselelo e-RNA.Ukunqanda iimveliso eziveliswe kwi-DNA ye-genomic, i-RNA inokunyangwa ngebakala lokukhulisa i-DnasⅠ ukususa i-DNA engcolisekileyo phambi kokuba kukhutshelwe umva.Iisampuli zigcinwe kwi-65℃ kwi-2.0mM EDTA ngemizuzu eyi-10 ukuphelisa ukugaya kwe-DNaseⅠ.I-EDTA chelates ion magnesium ukukhusela i-ion ye-magnesium exhomekeke kwi-RNA hydrolysis eyenzeka kumaqondo aphezulu.

Ukuze kwahlulwe i-cDNA eyandisiweyo kwimveliso yokukhulisa i-DNA ye-genome, iiprimers ezifakwa ngokwahlukeneyo nge-exon eyahluliweyo zinokuyilwa.Iimveliso ze-PCR ezivela kwi-cDNA ziya kuba mfutshane kunezo zivela kwi-DNA ye-genomic engcolileyo.Umfuniselo olawulwayo ngaphandle kokukhutshelwa umva wenziwa kwakhona kwitemplate nganye ye-RNA ukufumanisa ukuba iqhekeza elinikiweyo lisuka kwi-genomic DNA okanye i-cDNA.Iimveliso ze-PCR ezifunyenwe ngokungabikho kwe-reverse transcription ziphuma kwi-genome.

Imveliso eNxulumeneyo

RT-PCR Easy^ᵀᴹMna (Inyathelo elinye)

-Ikhithi yenyathelo elinye yenza ukuba uguqulelo lwe-reverse kunye ne-PCR iqhutywe kwi-tube efanayo.Ifuna kuphela ukongeza ithemplate ye-RNA, iiprimers ezithile zePCR kunye ne-RNase-Free ddH₂O.

-Uhlalutyo lwexesha langempela lobungakanani be-RNA lunokwenziwa ngokukhawuleza nangokuchanekileyo.

-Ikhithi isebenzisa i-reagent ekhethekileyo ye-Foregene reverse transcription reagent kunye ne-Foregene HotStar Taq DNA Polymerase idityaniswe nenkqubo yokusabela ekhethekileyo ukuze kuphuculwe ngokufanelekileyo ukusebenza kakuhle kokukhulisa kunye neenkcukacha zokuphendula.

-Inkqubo yokusabela ephuculweyo yenza i-reaction ibe novakalelo oluphezulu lokubona, ukuzinza okunamandla kwe-thermal, kunye nokunyamezela okungcono.

I-RT Easy II(Nge-GDNase) I-Master Premix ye-First-Strand CDNA Synthesis yeXesha lokwenyani PCR nge-GDNase

-Ikhono elisebenzayo lokususa i-gDNA, enokususa i-gDNA kwi-template ngaphakathi kwemizuzu emi-2.

-Inkqubo esebenzayo yokukhuphela umva, kuthatha imizuzu eyi-15 kuphela ukugqiba udityaniso lwe-cDNA yomtya wokuqala.

-Iitemplates eziyinkimbinkimbi: iitemplates ezinomxholo ophezulu weGC kunye nesakhiwo sesibini esiyinkimbinkimbi sinokuphinda sitshintshwe ngokusebenza okuphezulu.

-I-High-sensitivity reverse transcription system, i-pg-level templates inokufumana i-cDNA ephezulu.

-Isistim yokukhutshelwa umva inozinzo oluphezulu lwe-thermal, elona qondo lobushushu lokusabela liphezulu yi-42℃, kwaye isenomsebenzi omhle wokukhutshelwa umva kwi-50℃.