一, Mmụba uche nke usoro mmeghachi omume:

1. Wepụ RNA dị elu:

Ngwakọta cDNA na-aga nke ọma sitere na RNA dị elu.RNA dị elu kwesịrị opekata mpe bụrụ ogologo zuru oke yana enweghị ihe mgbochi transcriptase dị ka EDTA ma ọ bụ SDS.Ogo RNA na-ekpebi oke ozi usoro ị nwere ike idegharị na cDNA.Usoro RNA na-adị ọcha bụ otu nzọụkwụ na-eji guanidine isothiocyanate/acid phenol.Iji gbochie mmetọ site n'ịchọta ọnụọgụ RNase, RNA dịpụrụ adịpụ na nlele RNase bara ụba (dị ka pancreas) kwesịrị ịchekwa na formaldehyde iji chekwaa RNA dị elu, ọkachasị maka nchekwa ogologo oge.A na-emebisị RNA nke ewepụtara na imeju oke ka e debere ya na mmiri otu izu, ebe RNA nke ewepụtara na splin oke kwụsiri ike ka echekwara ya na mmiri afọ 3.Na mgbakwunye, transcript dị ogologo karịa 4 kb na-enwe mmetụta maka nbibi site na trace RNases karịa obere ederede.Iji mee ka nkwụsi ike nke ihe nlele RNA echekwara, enwere ike igbari RNA na formamide deionized wee chekwaa ya na -70°C.Formamide a na-eji echekwa RNA ga-enwerịrị ihe mkpori ndụ na-emebi RNA.Enwere ike ichekwa RNA sitere na pancreas na foramide opekata mpe otu afọ.Mgbe ị na-akwado iji RNA, ị nwere ike iji usoro na-esonụ iji kpalie RNA: tinye NaCl na 0.2M na 4 ugboro olu nke ethanol, tinye na ụlọ okpomọkụ maka 3-5 nkeji, na centrifuge na 10,000 × g maka 5 nkeji.

2. Jiri RNaseH-adịghị arụ ọrụ (RNaseH-) ntụgharị transcriptase:

A na-agbakwunyekarị ndị na-egbochi RNase ka ha weghachi mmeghachi omume ntụgharị ka iwelie ogologo na mkpụrụ nke njikọ cDNA.Ekwesịrị ịgbakwunye ndị na-egbochi RNase n'oge mmeghachi omume njikọ eriri nke mbụ n'ihu ihe nchekwa na onye na-ebelata ihe (dị ka DTT), n'ihi na usoro tupu njikọ cDNA na-eme ka onye na-emechi ihe na-eme ihe, si otú ahụ na-ahapụ RNase agbụ nke nwere ike imebi RNA.Ndị na-egbochi Protein RNase na-egbochi mmebi nke RNA site na RNase A, B, C, ma ghara igbochi RNase na akpụkpọ ahụ, ya mere kpachara anya ka ị ghara iwebata RNase site na mkpịsị aka gị n'agbanyeghị iji ihe mgbochi ndị a.

Reverse transcriptase na-eme ka ntụgharị RNA gaa cDNA.Ma M-MLV na AMV nwere ọrụ RNaseH endogenous na mgbakwunye na ọrụ polymerase nke ha.Ọrụ RNaseH na ọrụ polymerase na-asọrịta mpi n'otu n'otu maka eriri ngwakọ etolite n'etiti template RNA na DNA primer ma ọ bụ cDNA ndọtị, wee weda eriri RNA na RNA: mgbagwoju DNA.Ihe ndebiri RNA mebiri site na ọrụ RNaseH enweghịzi ike ije ozi dị ka mkpụrụ dị irè maka njikọ cDNA, nke na-ebelata mkpụrụ na ogologo nke njikọ cDNA.Ya mere, ọ ga-aba uru ikpochapụ ma ọ bụ belata nke ukwuu ọrụ RNaseH nke ntụgharị transcriptase..

SuperScript Ⅱ reverse transcriptase, RNaseH- MMLV reverse transcriptase na thermoScript reverse transcriptase, RNaseH-AMV, nwere ike nweta ego karịa na cDNA zuru oke karịa MMLV na AMV.Ọnụ ọgụgụ nke njikọ cDNA ga-emetụta uche RT-PCR.ThermoScript dị mmetụta karịa AMV.Oke ngwaahịa RT-PCR nwere oke site na ikike nke reverse transcriptase iji mepụta cDNA, ọkachasị mgbe ị na-emechi cDNA buru ibu.Tụnyere MMLV, SuperScripⅡ mụbara mkpụrụ nke ngwaahịa RT-PCR ogologo.RNaseH-reverse transcriptase nwekwara ụbara okpomọkụ, yabụ enwere ike ime mmeghachi omume na okpomọkụ dị elu karịa 37-42°C nkịtị.N'okpuru ọnọdụ njikọ akwadoro, jiri oligo(dT) primer na 10 μCi nke [α-P] dCTP.A gbakọọ mkpokọta mkpụrụ nke strand mbụ site na iji usoro iru mmiri TCA.A tụlere cDNA ogologo zuru oke site na iji bandị ndị nwere nha anya wepụrụ wee gụkọta ya na gel alkaline agarose.

3. Welie okpomọkụ incubation maka ntụgharị ntụgharị:

Okpomọkụ nnabata dị elu na-enyere aka imeghe usoro nke abụọ nke RNA, na-abawanye mkpụrụ nke mmeghachi omume.Maka ọtụtụ ndebiri RNA, incubating RNA na primers na 65ºC na-enweghị ihe nchekwa ma ọ bụ nnu, na-esote ngwa ngwa oyi na ice ga-ewepụ ọtụtụ ụlọ nke abụọ ma kwe ka primers kekọta.Agbanyeghị, ụfọdụ ndebiri ka nwere usoro nke abụọ, ọbụlagodi mgbe denaturation okpomọkụ gasịrị.Enwere ike ime nkwalite ndebiri ndị a siri ike site na iji ThermoScript Reverse Transcriptase na idowe mmeghachi omume ntụgharị na okpomọkụ dị elu iji melite nkwalite.Okpomọkụ nnabata dị elu nwekwara ike ịbawanye nkọwapụta, ọkachasị mgbe ejiri mkpụrụ ndụ ihe nketa-kpọmkwem (GSP) maka njikọ cDNA (lee Isi nke 3).Ọ bụrụ na ị na-eji GSP, hụ na Tm nke primers bụ otu ihe ahụ dị ka okpomọkụ incubation a na-atụ anya ya.Ejila oligo(dT) na primers na-enweghị ihe karịrị 60°C.Ndị na-emepụta ihe na-achọghị ka a na-etinye ya na 25 Celsius C maka nkeji 10 tupu ha eruo 60 Celsius.Na mgbakwunye na iji okpomọkụ ntụgharị dị elu dị elu, a pụkwara imeziwanye nkọwapụta site na ịnyefe ngwakọta RNA / primer ozugbo site na 65 Celsius C denaturation okpomọkụ na ntụgharị transcription incubation okpomọkụ na ịgbakwunye ngwakọta mmeghachi omume 2 × tupu ọkụ (cDNA hot-mmalite njikọ) .Ụzọ a na-enyere aka igbochi njikọ njikọ intermolecular base nke na-eme na obere okpomọkụ.Enwere ike ime ka ngbanwe nke otutu okpomọkụ achọrọ maka RT-PCR site na iji igwe igwe ọkụ.

polymerase Tth thermostable na-arụ ọrụ dị ka DNA polymerase n'ihu Mg2+ yana dị ka RNA polymerase n'ihu Mn2+.Enwere ike idobe ya ọkụ na oke okpomọkụ nke 65 Celsius.Otú ọ dị, ọnụnọ Mn2 + n'oge PCR na-ebelata iguzosi ike n'ihe, nke na-eme ka Tth polymerase ghara ịdị mma maka mmụba dị elu, dị ka cloning nke cDNA.Tụkwasị na nke ahụ, Tth nwere arụmọrụ ntụgharị ntụgharị dị ala, nke na-ebelata uche, na, ebe ọ bụ na ntụgharị ntụgharị na PCR nwere ike iji otu enzyme mee ihe, mmeghachi omume nchịkwa na-enweghị ntụgharị ntụgharị enweghị ike iji tụnyere ngwaahịa mmụba cDNA na DNA genomic na-emerụ emerụ.E kewapụrụ ngwaahịa nkwalite ahụ.

4. Ihe mgbakwunye na-akwalite ntụgharị ntụgharị:

A na-agbakwunye ihe mgbakwunye gụnyere glycerol na DMSO na mmeghachi omume njikọ nke mbụ, nke nwere ike belata nkwụsi ike nke nucleic acid okpukpu abụọ ma tọpụ usoro nke abụọ nke RNA.Enwere ike itinye ihe ruru 20% glycerol ma ọ bụ 10% DMSO na-emetụtaghị ọrụ SuperScript II ma ọ bụ MMLV.AMV nwekwara ike anabata ihe ruru 20% glycerol na-enweghị mfu nke ọrụ.Ka iwelie nghọta nke RT-PCR na SuperScriptⅡ mmeghachi omume ntụgharị ntụgharị, enwere ike ịgbakwunye 10% glycerol ma tinye ya na 45°C.Ọ bụrụ na 1/10 nke ngwaahịa mmeghachi omume reverse transcription agbakwunyere na PCR, mgbe ahụ ntinye nke glycerol na mmeghachi omume mmụba bụ 0.4%, nke ezughị iji gbochie PCR.

5. ọgwụgwọ RNaseH:

Ọgwụgwọ mmeghachi omume cDNA na RNaseH tupu PCR nwere ike ịbawanye nghọta.Maka ụfọdụ ndebiri, a na-eche na RNA na mmeghachi omume cDNA na-egbochi njikọ nke ngwaahịa mmụba, nke ọgwụgwọ RNaseH nwere ike ịbawanye uche.N'ozuzu, ọgwụgwọ RNaseH dị mkpa mgbe ị na-eme ka ndebiri cDNA dị ogologo zuru oke, dị ka obere oyiri tuberous scherosis II.Maka ndebiri a siri ike, ọgwụgwọ RNaseH kwalitere mgbama nke SuperScript II ma ọ bụ cDNA arụnyere AMV mepụtara.Maka ọtụtụ mmeghachi omume RT-PCR, ọgwụgwọ RNaseH bụ nhọrọ, n'ihi na nkwụsị PCR na 95 Celsius na-emekarị hydrolyzes RNA na RNA: DNA complex.

6. Mmelite nke obere ụzọ nchọpụta RNA:

RT-PCR bụ ihe ịma aka karịsịa mgbe naanị obere RNA dị.Glycogen agbakwunyere dị ka onye na-ebu n'oge ikewapụ RNA na-enyere aka ịbawanye mkpụrụ nke obere nlele.Enwere ike ịgbakwunye glycogen na-enweghị RNase n'otu oge yana ịgbakwunye Trizol.Glycogen bụ mmiri na-agbaze ma nwee ike idowe ya n'oge mmiri mmiri na RNA iji nyere aka mmiri ozuzo na-esote.Maka ihe nlele na-erughị 50 mg nke anụ ahụ ma ọ bụ sel 106 omenala, ntinye glycogen na-enweghị RNase akwadoro bụ 250 μg/ml.

Ịgbakwunye BSA acetylated na mmeghachi omume ntụgharị ntụgharị site na iji SuperScript II nwere ike ịbawanye nghọta, yana maka obere RNA, ibelata ọnụọgụ SuperScript II yana ịgbakwunye nkeji 40 nke ihe mgbochi nuclease RNaseOut nwere ike ịbawanye ọkwa nke nchọpụta.Ọ bụrụ na ejiri glycogen na usoro ikewapụ RNA, a ka na-atụ aro ka ịgbakwunye BSA ma ọ bụ RNase inhibitor mgbe ị na-eji SuperScript II maka mmeghachi omume ntụgharị ntụgharị.

Dịkwuo, Mụbaa kpọmkwem RT-PCR

1. CND njikọ:

Enwere ike ibido njikọ cDNA nke mbụ site na iji ụzọ atọ dị iche iche, nkọwa nkọwa ya na-emetụta ọnụọgụ na ụdị cDNA synthesized.

Usoro primer random bụ nke kacha nta kpọmkwem n'ime ụzọ atọ ahụ.Primers na-akpachapụ n'ọtụtụ saịtị n'ime transcript a, na-ewepụta cDNA dị mkpụmkpụ, ogologo akụkụ.A na-ejikarị usoro a nweta usoro njedebe 5' yana nweta cDNA site na ndebiri RNA nwere mpaghara nhazi nke abụọ ma ọ bụ saịtị nkwụsị nke enweghị ike ịmegharị ya site na ntụgharị transcriptase.Iji nweta cDNA kachasị ogologo, oke nke primers na RNA dị na nlele RNA ọ bụla kwesịrị ikpebi nke ọma.Nleba anya mmalite nke primers random sitere na 50 ruo 250 ng kwa mmeghachi omume 20 μl.Ebe ọ bụ na cDNA arụpụtara site na mkpokọta RNA site na iji primers random bụ RNA ribosomal, a na-ahọrọ poly(A)+ RNA dị ka ndebiri.

Oligo(dT) primers a kapịrị ọnụ karịa ngwa ngwa ngwa.Ọ na-ejikọta ọnụ ruo ọdụ poly(A) achọtara na njedebe 3′ nke ọtụtụ mRNA eukaryotic.N'ihi na poly(A)+ RNA dị ihe dị ka 1% ruo 2% nke mkpokọta RNA, ego na mgbagwoju anya nke cDNA dị obere karịa ka ọ na-eji primers enweghị usoro.N'ihi nkọwapụta ya dị elu, oligo(dT) anaghị achọ nkwalite oke nke RNA na primers yana nhọrọ poly(A)+.A na-atụ aro ka iji 0.5μg oligo(dT) kwa 20μl mmeghachi omume.oligo (dT) 12-18 dabara maka ọtụtụ RT-PCR.ThermoScript RT-PCR Sistemu na-enye oligo(dT)20 n'ihi nkwụsi ike ya kacha mma maka okpomọkụ incubation dị elu.

Gene Specific primers (GSP) bụ ihe izizi izizi maka ntụgharị ntụgharị.GSP bụ oligonucleotide antisense nke nwere ike ịgbakọ kpọmkwem n'usoro ebumnuche RNA, n'adịghị ka primers na-enweghị usoro ma ọ bụ oligo(dT), nke na-akụda RNA niile.Otu iwu a na-eji chepụta primers PCR na-emetụta imewe nke GSP na mmeghachi omume ntụgharị ntụgharị.GSP nwere ike ịbụ otu usoro ahụ dị ka primer mmụba nke na-agbada na 3′-kasị njedebe nke mRNA, ma ọ bụ GSP nwere ike ime ka ọ kwụsịlata ala nke primer ntụgharị.Maka ụfọdụ isiokwu agbakwunyere, ọ dị mkpa ka emebe ihe karịrị otu primer antisense maka RT-PCR na-aga nke ọma n'ihi na nhazi nke abụọ nke RNA ebumnuche nwere ike igbochi njikọ nke primer.A na-atụ aro ka iji 1 pmol antisense GSP na mmeghachi omume njikọ eriri mbụ nke 20 μl.

2. Welie okpomọkụ incubation maka ntụgharị ntụgharị:

Iji nweta uru zuru oke nke nkọwa GSP, a ga-eji ntụgharị transcriptase nwere okpomọkụ dị elu.Enwere ike itinye transcriptases reverse thermostable na okpomọkụ dị elu ka iwelie eriri mmeghachi omume.Dịka ọmụmaatụ, ọ bụrụ na GSP anneals na 55°C, a gaghị eji ya arụ ọrụ nke ọma nke GSP ma ọ bụrụ na ejiri AMV ma ọ bụ M-MLV mee ntụgharị ntụgharị na obere eriri 37°C.Otú ọ dị, SuperScript II na ThermoScript nwere ike imeghachi omume na 50°C ma ọ bụ karịa, nke ga-ewepụ ngwaahịa ndị na-abụghị nke a na-emepụta na obere okpomọkụ.Maka nkọwapụta kachasị, enwere ike ibufe ngwakọta RNA/primer ozugbo site na okpomoku denaturation 65°C gaa na ntụgharị ntụgharị ntụgharị ma tinye ya na ngwakọta mmeghachi omume 2 × tupu ọkụ (njikọ cDNA na-ekpo ọkụ).Nke a na-enyere aka igbochi njikọta intermolecular base na obere okpomọkụ.Enwere ike ime ka ọtụtụ mgbanwe okpomọkụ achọrọ maka RT-PCR site na iji igwe igwe na-ekpo ọkụ.

3. Na-ebelata mmetọ DNA genomic:

Ihe isi ike nwere ike izute na RT-PCR bụ mmetọ nke DNA genomic na RNA.Iji ezigbo RNA kewapụrụ onwe ya, dị ka Trizol Reagent, ga-ebelata ọnụọgụ DNA genomic na-emerụ nkwadebe RNA.Iji zere ngwaahịa ndị sitere na DNA genomic, enwere ike ịgwọ RNA na mmụba-ọkwa DNase I iji wepụ DNA na-emerụ emerụ tupu iweghachi ya.Akwụsịla mgbaze DNAse I site n'ịkwanye ihe nlele ahụ na 2.0 mM EDTA maka nkeji 10 na 65°C.EDTA nwere ike chelate ion magnesium, na-egbochi magnesium ion dabere na RNA hydrolysis na oke okpomọkụ.

Iji kewapụ cDNA agbakwunyere na ngwaahịa mmụba DNA na-emerụ emerụ, enwere ike ịhazi primers ka onye ọ bụla na-ewepụ ikewapụ exons.Ngwaahịa PCR ewepụtara na cDNA ga-adị mkpụmkpụ karịa ndị ewepụtara site na DNA genomic mmeru.Na mgbakwunye, a na-eme nnwale njikwa na-enweghị ntụgharị ntụgharị na ndebiri RNA nke ọ bụla iji chọpụta ma e nwetara iberibe e nyere site na DNA genomic ma ọ bụ cDNA.Ngwaahịa PCR enwetara na-enweghị ntụgharị ntụgharị sitere na genome.