Ⅰ. Mee ka uche nke sistemu mmeghachi omume dịkwuo elu:

1. RNA dị elu dị iche iche:

Ngwakọta cDNA na-aga nke ọma sitere na RNA dị elu.RNA dị elu kwesịrị ịhụ na opekata mpe ogologo oge na enweghị ihe mgbochi na-enweghị enzymes ndekọ, dị ka EDTA ma ọ bụ SDS.Ogo RNA na-ekpebi uru kachasị nke ozi usoro ị nwere ike ịdegharị na cDNA.Usoro nchacha nke RNA izugbe bụ usoro nzọụkwụ nke iji isoocyanate/acidophenol.Iji gbochie mmetọ nke RNase, RNA kewapụrụ na ihe atụ bara ụba na RNase (dị ka pancreas) chọrọ nchekwa nke formaldehyde iji chekwaa RNA dị elu, nke na-adịkwa karịa maka nchekwa ogologo oge.A na-emebisị RNA nke ewepụtara na imeju oke ka otu izu nke nchekwa n'ime mmiri gachara, ebe RNA ewepụtara na splin oke ahụ kwụsiri ike mgbe afọ atọ nke nchekwa na mmiri gasịrị.Na mgbakwunye, transcripts buru ibu karịa 4kb na-enwe mmetụta nke ọma maka ịchọpụta mmebi RNase karịa obere ndegharị.Iji mee ka nkwụsi ike nke ihe nlele RNA nchekwa, enwere ike ịgbaze RNA na methalmamine nke ion, ma chekwaa -70 Celsius.Thylide eji chekwaa RNA agaghị enwe ihe dị iche iche na-eweda RNA.Enwere ike ịchekwa RNA, nke sitere na pancreas, na methalmamine ma ọ dịkarịa ala otu afọ.Mgbe ị dị njikere iji RNA, ị nwere ike iji ụzọ ndị a iji mee ka RNA na-ebuli: tinye NaCl na 0.2m na 4 ugboro olu nke ethanol, tinye okpomọkụ ụlọ maka nkeji 3-5, na 10,000 × g centrifugal maka nkeji ise.

2. Jiri reverse transcriptase na-enweghị ọrụ RNaseH (RNaseH-):

A na-agbakwunyekarị ndị na-egbochi RNase ka ha weghachi mmeghachi omume ntụgharị ka iwelie ogologo na mkpụrụ nke njikọ cDNA.A na-agbakwunye RNase inhibitor na mmeghachi omume njikọ agbụ nke mbụ n'ihu ndị na-echekwa ma na-ebelata ihe ndị dị ka DTT n'ihi na tupu cDNA synthesis usoro denatures inhibitor, si otú na-ahapụ bounded RNases na-eweda RNA.Protein RNase inhibitor na-egbochi mmebi nke RNA site na RNase A, B, C, ma ghara igbochi RNases na akpụkpọ ahụ, ya mere ekwesịrị ịkpachara anya ka ị ghara iwebata RNase site na mkpịsị aka n'agbanyeghị iji ihe mgbochi ndị a.

Reverse transcriptase na-eme ka ntụgharị RNA bụrụ cDNA.Ma M-MLV na AMV nwere ọrụ RNaseH endogenous na mgbakwunye na ọrụ polymerase nke ha.Ọrụ RNaseH na-asọ mpi polymerase maka eriri heterozygous kpụrụ n'etiti ndebiri RNA na primers DNA ma ọ bụ eriri ndọtị cDNA, na-eweda RNA: eriri RNA na ogige DNA.A gaghịzi eji ndebiri RNA mebiri site na ọrụ RNaseH dị ka ihe nrụpụta dị irè maka njikọ cDNA, na-ebelata mkpụrụ na ogologo njikọ cDNA.Ya mere iwepụ ma ọ bụ ibelata nke ukwuu ọrụ RNaseH nke reverse transcriptase ga-aba uru dị ukwuu.

SuperScriptⅡ reverse transcriptase, MMLV reverse transcriptase nke RNaseH- na thermoScript reverse transcriptase, AMV nke RNaseH- wepụtara cDNA n'ogologo karịa MMLV na AMV.Ọnụ ọgụgụ cDNA arụpụtara na-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 Cdnas buru ibu.Tụnyere MMLV, SuperScripⅡ mụbara mkpụrụ nke ngwaahịa RT-PCR ogologo.RNaseH-'s reverse transcriptase na-abawanye nkwụsi ike nke okpomọkụ, yabụ enwere ike ịme mmeghachi omume na okpomọkụ dị elu karịa nke 37-42 ℃.N'okpuru ọnọdụ njikọ akwadoro, a na-eji oligo (dT) primers na 10μCi [alpha-p] dCTP mee ihe.A gbakọọ mkpokọta mmepụta nke yinye mbụ site na iji usoro mmiri ozuzo TCA.A tụlere cDNA ogologo zuru oke site na iji mwepụ nke nha nha na agụta na gel alkaline agarose.

3. Dịkwuo okpomọkụ ichekwa okpomọkụ nke reverse transcription:

Okpomọkụ njide dị elu na-enyere aka imepe usoro nke abụọ nke RNA ma mụbaa mkpụrụ nke mmeghachi omume.Maka ọtụtụ ndebiri RNA, ijide RNA na primer na 65ºC na-enweghị ihe nchekwa ma ọ bụ nnu wee mee ka ha dị jụụ ngwa ngwa na ice na-ewepụ ọtụtụ ụlọ nke abụọ ma na-enye ohere ka ejikọta primers.Agbanyeghị, ụfọdụ ndebiri ka nwere nhazi nke abụọ, ọbụlagodi mgbe denaturation thermal gachara.Enwere ike ime nkwalite ndebiri ndị a siri ike site na iji ThermoScript reverse transcriptase yana site n'itinye mmeghachi omume transcriptase na okpomọkụ dị elu iji melite nkwalite.Okpomọkụ njide dị elu nwekwara ike ịbawanye nkọwapụta, ọkachasị mgbe a na-eme njikọ cDNA site na iji mkpụrụ ndụ ihe dị iche iche (GSPS) (lee Isi nke 3).Ọ bụrụ na ị na-eji GSP, jide n'aka na uru Tm nke primer bụ otu ihe ahụ dị ka okpomọkụ a na-atụ anya ya.Ejila oligo(dT) na primers random n'elu 60 ℃.Random primers kwesịrị ka ejidere na 25 ℃ maka nkeji 10 tupu abawanye ruo 60 ℃.Na mgbakwunye na iji okpomọkụ ntụgharị ntụgharị dị elu, enwere ike imeziwanye nkọwapụta site na ịnyefe ngwakọta RNA / primer ozugbo site na 65 ℃ denaturing okpomọkụ na ntụgharị ntụgharị na-ejide okpomọkụ yana ịgbakwunye ngwakọta mmeghachi omume 2 × preheated (cDNA thermal initiation synthesis).Ụzọ a na-enyere aka igbochi njikọ njikọ intermolecular base nke na-eme na obere okpomọkụ.Iji ngwa PCR na-eme ka ọtụtụ mgbanwe ọkụ chọrọ maka RT-PCR dị mfe.

Tth okpomọkụ kwụsiri ike polymerase na-arụ ọrụ dị ka DNA polymerase n'ihu Mg2+ na RNA polymerase n'ihu Mn2+.Ọ nwere ike ijide okpomọkụ ruo 65 ℃.Otú ọ dị, ọnụnọ Mn2 + n'oge PCR na-ebelata ikwesị ntụkwasị obi, nke na-eme ka Tth polymerase ghara ịdị mma maka mmụba dị elu, dị ka cDNA cloning.Tụkwasị na nke ahụ, Tth adịghị arụ ọrụ nke ọma na ntụgharị ntụgharị, nke na-ebelata uche, na ebe ọ bụ na otu enzyme nwere ike ime ntụgharị ntụgharị na PCR, mmeghachi omume nchịkwa na-enweghị ntụgharị ntụgharị enweghị ike iji chọpụta ngwaahịa cDNA gbasiri ike na nke DNA genomic merụọ.

4. mgbakwunye na-akwalite ntụgharị ntụgharị:

Mgbakwunye nke ihe mgbakwunye, gụnyere glycerin na DMSO, na njikọ njikọ nke mbụ nwere ike ibelata nkwụsi ike nke eriri nucleic acid okpukpu abụọ ma weghachi usoro nke abụọ RNA.Enwere ike itinye ihe ruru 20% glycerin ma ọ bụ 10% DMSO na-emetụtaghị ọrụ SuperScriptⅡ ma ọ bụ MMLV.AMV nwekwara ike anabata ihe ruru 20% glycerol na-enweghị ibelata ọrụ.Iji bulie nghọta nke RT-PCR na SuperScriptⅡ mmeghachi omume ntụgharị ntụgharị, enwere ike ịgbakwunye 10% glycerol na mkpuchi na 45 ℃.Ọ bụrụ na 1/10 nke ngwaahịa retrotranscription-reaction agbakwunyere na PCR, ntinye nke glycerol na mmeghachi omume mmụba bụ 0.4%, nke ezughị iji gbochie PCR.

5. RNaseH nhazi:

Enwere ike imeziwanye uche site n'ịgwọ mmeghachi omume njikọ cDNA na RNaseH tupu PCR.Maka ụfọdụ ndebiri, a na-eche na RNA na mmeghachi omume cDNA na-egbochi njikọ nke ngwaahịa ndị agbakwunyere, nke ikpe RNaseH nwere ike ịbawanye uche.N'ozuzu, a chọrọ ọgwụgwọ RNaseH maka mmụba nke ụdị cDNA lekwasịrị anya ogologo ogologo, dị ka tuberous scherosisⅡ nwere obere oyiri.Maka ndebiri a siri ike, RNaseH kwalitere mgbama sitere na cDNA nke SuperScriptⅡ ma ọ bụ AMV mebere.Maka ọtụtụ mmeghachi omume RT-PCR, ọgwụgwọ RNaseH bụ nhọrọ n'ihi na 95 ℃ mkpọchi PCR denaturation nzọụkwụ na-emekarị ka RNA si na RNA: mgbagwoju DNA.

6. Ụzọ emelitere maka ịchọpụta obere RNA:

RT-PCR bụ ihe ịma aka karịsịa mgbe naanị obere RNA dị.Mgbakwunye glycogen dị ka onye na-ebu n'oge nkewa RNA na-enyere aka ịbawanye mkpụrụ nke obere ihe atụ.Enwere ike ịgbakwunye glycogen na-enweghị RNase n'otu oge yana Trizol.Glycogen bụ mmiri na-agbaze ma nwee ike ịnọgide na mpaghara mmiri yana RNA iji nyere aka na mmiri ozuzo na-esote.Ntinye uche nke glycogen na-enweghị RNase bụ 250μg/ml maka nlele na-erughị 50mg nke anụ ahụ ma ọ bụ sel 106 omenala.

Mgbakwunye nke BSA acetylated iji tụgharịa mmeghachi omume ntụgharị site na iji SuperScriptⅡ nwere ike ịbawanye nghọta, yana maka obere RNA, ibelata ọnụọgụ SuperScriptⅡ yana ịgbakwunye nkeji 40 nke RnaseOut nuclease inhibitor nwere ike melite ọkwa nchọpụta.Ọ bụrụ na ejiri glycogen na nkewa RNA, mgbakwunye BSA ma ọ bụ RNase inhibitors ka akwadoro mmeghachi omume ntụgharị site na iji SuperScriptⅡ.

Ⅱ. Mụbaa nkọwapụta nke RT-PCR

1. njikọ cNDA:

Enwere ike iji ụzọ atọ dị iche iche wee malite njikọ cDNA strand nke mbụ, na nkọwa nkọwa nke usoro ọ bụla na-emetụta ọnụọgụ na ụdị cDNA ahazi.

Usoro primer random bụ nke kacha nta kpọmkwem n'ime ụzọ atọ a.A na-ekpochapụ primers n'ọtụtụ saịtị n'ime ederede niile iji mepụta cDNA dị mkpụmkpụ, ogologo akụkụ.A na-ejikarị usoro a nweta usoro njedebe 5′ yana cDNA site na ndebiri RNA nwere mpaghara nhazi nke abụọ ma ọ bụ nwere saịtị nkwụsị nke na-atụgharị transcriptase enweghị ike ịmegharị.Iji nweta cDNA kachasị ogologo, oke nke primers na RNA dị na nlele RNA ọ bụla kwesịrị ikpebi nke ọma.Ntinye uche izizi nke primers random sitere na 50 ruo 250ng kwa sistemu mmeghachi omume 20μl.N'ihi na cDNA arụpụtara site na mkpokọta RNA site na iji primers random bụ nke kachasị RNA ribosomal, a na-ahọrọ poly(A)+ RNA dị ka ndebiri.

Mmalite Oligo(dT) akọwapụtara nke ọma karịa ihe mbido enweghị usoro.Ọ na-ejikọta ya na ọdụ poly(A) achọtara na njedebe 3′ nke mRNA n'ime ọtụtụ sel 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 ma ọ bụrụ na ejiri ihe na-enweghị usoro.N'ihi nkọwapụta ya dị elu, oligo(dT) anaghị achọ njikarịcha maka RNA ka primer ratio na poly(A)+ nhọrọ.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 Sistemụ na-enye oligo(dT)20 n'ihi nkwụsi ike ya dị mma ma dabara adaba maka ọnọdụ okpomọkụ dị elu.

Gene-specific primers (GSP) bụ ihe izizi kacha mma maka ntụgharị ntụgharị.GSP bụ oligonucleoside antisense nke nwere ike iji usoro ebe njedebe RNA jikọtara ọnụ, kama ikpochapụ Rna niile dịka primers random ma ọ bụ oligo(dT).Iwu ndị a na-eji chepụta primers PCR na-emetụtakwa imewe mmeghachi omume ntụgharị ntụgharị GSP.GSP nwere ike ịbụ otu usoro dị ka primer amplification annealed na njedebe nke mRNA3′, ma ọ bụ GSP nwere ike ime ka anneatị na mgbada ala site na iji primer ntụgharị ntụgharị.Maka ụfọdụ ihe agbakwunyere, ọ dị mkpa ịmepụta ihe karịrị otu antisense primer maka RT-PCR na-aga nke ọma n'ihi na nhazi nke abụọ nke RNA ebumnuche nwere ike igbochi primer ka ọ ghara ijikọ.A na-atụ aro ka iji 1pmol antisense GSP na usoro mmeghachi omume nke mbụ nke 20μl.

2. Dịkwuo okpomọkụ ichekwa okpomọkụ nke reverse transcription:

Iji nweta uru zuru oke nke nkọwa GSP, tụgharịa transcriptase nwere nkwụsi ike dị elu kwesịrị iji.Enwere ike idobe transcriptase reverse nke na-ekpo ọkụ na okpomọkụ dị elu iji mee ka mmeghachi omume siri ike.Dịka ọmụmaatụ, ọ bụrụ na ekpochapụ GSP na 55ºC, mgbe ahụ, a naghị eji kpọmkwem GSP arụ ọrụ ma ọ bụrụ na a na-eme ntụgharị ntụgharị na 37 ° C na obere rigor site na iji AMV ma ọ bụ M-MLV.Agbanyeghị, SuperScripⅡ na ThermoScript nwere ike meghachi omume na 50℃ ma ọ bụ karịa, nke na-ewepụ ngwaahịa na-abụghị nke a na-emepụta na obere okpomọkụ.Maka nkọwa kachasị, enwere ike ibufe ngwakọta RNA/ primer ozugbo site na 65 ℃ denaturation okpomọkụ gaa na ntụgharị ederede na-ejide okpomọkụ yana mgbakwunye nke ngwakọta mmeghachi omume 2 x ekpo ọkụ (mmalite okpomọkụ nke cDNA njikọ).Nke a na-enyere aka igbochi njikọ ntọala n'etiti ụmụ irighiri ihe na obere okpomọkụ.Iji ngwa PCR na-eme ka ọ dị mfe ọtụtụ mgbanwe okpomọkụ achọrọ maka RT-PCR.

3. Belata mmetọ DNA nke genomic:

Otu ihe isi ike nwere ike na RT-PCR bụ na RNA na-emetọ DNA genomic.Iji ụzọ nkewa RNA ka mma, dị ka Trizol Reagent, na-ebelata mmetọ DNA genomic na nkwadebe RNA.Iji zere ngwaahịa ndị sitere na DNA genomic, enwere ike ịgwọ RNA na ọkwa nkwalite DNA iji wepụ DNA emetọọ tupu e degharịa ya.Edebere ihe nlele ahụ na 65℃ na 2.0mM EDTA maka nkeji 10 iji kwụsị mgbaze DNAseⅠ.EDTA chelates magnesium ion iji gbochie magnesium ion dabere RNA hydrolysis nke na-eme na oke okpomọkụ.

Iji kewapụ cDNA agbagoro na ngwaahịa genome DNA amplification, enwere ike ịmepụta primers nke na-ewepụ iche na exon kewapụrụ.Ngwaahịa PCR ewepụtara na cDNA ga-adị mkpụmkpụ karịa ndị ewepụtara site na DNA genomic mmeru.A na-eme nnwale a na-achịkwa na-enweghị ntụgharị ntụgharị na ndebiri RNA ọ bụla iji chọpụta ma iberibe e nyere sitere na DNA genomic ma ọ bụ cDNA.Ngwaahịa PCR enwetara na enweghị ntụgharị ntụgharị sitere na genome.

Ngwaahịa emetụtara

RT-PCR dị mfe^ᵀᴹM (Otu Nzọụkwụ)

- Ngwa otu nzọụkwụ na-enyere aka ntụgharị ntụgharị na PCR na-eme n'otu tube.Naanị ọ ga-achọ ịgbakwunye RNA template, kpọmkwem PCR primers na RNase-Free ddH₂O.

-Enwere ike ime nyocha ọnụọgụ nke RNA ozugbo na ngwa ngwa.

Ngwa ahụ na-eji ihe ntụgharị ntụgharị pụrụ iche nke Foregene na Foregene HotStar Taq DNA Polymerase jikọtara ya na sistemu mmeghachi omume pụrụ iche iji melite arụmọrụ nkwalite na nkọwapụta nke mmeghachi omume ahụ nke ọma.

-Usoro mmeghachi omume kachasị mma na-eme ka mmeghachi omume ahụ nwee mmetụta nchọpụta dị elu, nkwụsi ike okpomọkụ siri ike, na ndidi ka mma.

RT Easy II (ya na GDNase) Master Premix maka CDNA Synthesis nke mbụ maka PCR na GDNase

-Ike dị mma iwepu gDNA, nke nwere ike wepu gDNA na ndebiri n'ime nkeji 2.

Sistemụ ntụgharị ntụgharị nke ọma, ọ na-ewe naanị nkeji iri na ise iji mechaa njikọ nke eriri cDNA mbụ.

-Edebere mgbagwoju anya: ndebiri nwere ọdịnaya GC dị elu yana usoro nke abụọ dị mgbagwoju anya nwekwara ike iji ọrụ dị elu tụgharịa.

Sistemụ ntụgharị ntụgharị uche dị elu, ndebiri pg nwere ike nweta cDNA dị elu.

Sistemụ ntụgharị ntụgharị nwere nkwụsi ike dị elu, ezigbo mmeghachi omume okpomọkụ bụ 42 ℃, ọ ka nwere arụmọrụ ntụgharị ntụgharị dị mma na 50 ℃.