Emepụtara RT-qPCR site na teknụzụ PCR nkịtị.Ọ na-agbakwunye kemịkalụ fluorescent (ụcha fluorescent ma ọ bụ nyocha fluorescent) na sistemu mmeghachi omume PCR ọdịnala, wee chọpụta usoro mbelata PCR na ndọtị ozugbo dị ka usoro luminescent ha dị iche iche si dị.A na-eji mgbanwe mgbama fluorescent na ọkara iji gbakọọ ọnụọgụ mgbanwe ngwaahịa na okirikiri nke PCR ọ bụla.Ka ọ dị ugbu a, ụzọ ndị a na-ahụkarị bụ usoro agba agba fluorescent na usoro nyocha.

Usoro agba agba fluorescent:
Ụfọdụ ihe eji esiji fluorescent, dị ka SYBR Green Ⅰ, PicoGreen, BEBO, wdg, anaghị amụnye ọkụ n'onwe ha, kama na-ebunye fluorescence mgbe ejikọtara ya na obere oghere nke dsDNA.Ya mere, na mmalite nke mmeghachi omume PCR, igwe enweghị ike ịchọpụta mgbaàmà fluorescent.Mgbe mmeghachi omume na-aga n'ihu na mgbatị ahụ-mgbatị (usoro nzọụkwụ abụọ) ma ọ bụ mgbatị mgbatị (usoro nzọụkwụ atọ), a na-emeghe eriri abụọ ahụ n'oge a, na DNA polymerase ọhụrụ N'oge njikọ eriri, a na-ejikọta molecules fluorescent na dsDNA obere groove na emit fluorescence.Ka ọnụ ọgụgụ nke okirikiri PCR na-abawanye, ọtụtụ ihe eji esijiji na-ejikọta na dsDNA, na mgbama fluorescent na-aga n'ihu na-abawanye.Were SYBR Green Ⅰ dị ka ọmụmaatụ.
Usoro nyocha:
Nyocha Taqman bụ nyocha hydrolysis nke a na-ejikarị eme ihe.Enwere otu fluorescent na njedebe 5′ nke nyocha, na-abụkarị FAM.Nyocha n'onwe ya bụ usoro na-akwado mkpụrụ ndụ ihe nketa.Enwere otu ndị na-emenyụ fluorescent na njedebe 3 nke fluorophore.Dị ka ụkpụrụ nke fluorescence resonance ike nyefe (Förster resonance energy transfer, FRET), mgbe onye nta akụkọ fluorescent otu (enye onyinye fluorescent molekul) na quenching fluorescent otu (nabata fluorescent molecule) Mgbe excitation ụdịdị dị iche iche overlaps na anya dị nnọọ nso (7-10nm nke nwere ike na-anabata na excimole). molecule, ebe autofluorescence na-ebelata.Ya mere, na mmalite nke mmeghachi omume PCR, mgbe nyocha ahụ nwere onwe ya na nke na-adịghị mma na usoro ahụ, ndị nta akụkọ fluorescent otu agaghị eme ka ọ pụta ìhè.Mgbe ị na-ehichapụ, primer na nyocha na-ejikọta na template.N'oge mgbatị ahụ, polymerase na-ejikọta ụdọ ọhụrụ.DNA polymerase nwere 5′-3′ exonuclease ọrụ.Mgbe ị na-erute nyocha ahụ, DNA polymerase ga-eme ka nyocha ahụ dị na template, kewapụ ndị nta akụkọ fluorescent otu na otu quencher fluorescent otu, ma hapụ akara ngosi fluorescent.Ebe ọ bụ na enwere mmekọrịta n'otu n'otu n'etiti nyocha na template, usoro nyocha dị elu karịa usoro agba agba n'ihe ziri ezi na nghọta nke ule.

Isi 1 ụkpụrụ nke qRT-PCR

Nhazi mbụ
Ụkpụrụ:

Ekwesịrị ịhazi primers na mpaghara echekwara nke usoro nucleic acid ma nwee nkọwa.

Ọ kacha mma iji usoro cDNA, na usoro mRNA bụkwa nke a na-anabata.Ọ bụrụ na ọ bụghị, chọpụta nhazi mpaghara cds nke usoro DNA.
Ogologo ngwaahịa nke fluorescent quantitative bụ 80-150bp, nke kachasị ogologo bụ 300bp, ogologo nke primer bụ n'ozuzu n'etiti 17-25 bases, na ọdịiche dị n'etiti elu na ala primers ekwesịghị ịdị ukwuu.

Ọdịnaya G+C dị n'etiti 40% na 60%, yana 45-55% kacha mma.
Uru TM dị n'etiti ogo 58-62.
Gbalịa izere primer dimers na self-dimers, (adịghị apụta karịa 4 ụzọ abụọ nke consecutive nkwado bases) hairpin Ọdịdị, ma ọ bụrụ na-apụghị izere ezere, mee ΔG <4.5kJ / mol * Ọ bụrụ na ị na-apụghị hụ na gDNA e wepụrụ n'oge reverse transcription Dị ọcha, ọ kasị mma ịmepụta primers nke intron, ghara ime mgbanwe G , T / G, na-ezere mpaghara mpaghara * 3 'G' usoro na-aga n'ihu (2-3) primers na ndị na-abụghị.
kpọmkwem The homology nke heterogeneously amụba usoro bụ ọkachamma na-erughị 70% ma ọ bụ nwere 8 nkwado ndabere homology.
Ebe nchekwa data:
Chọọ OwuFGD site na mkpụrụokwu
Nhazi izizi:
IDT-qPCR imewe

Fig2 IDT online primer imewe ibe

Ngosipụta ibe nsonaazụ Fig3
Nhazi nke primers lncRNA:
lncRNA:Otu usoro dị ka mRNA.
miRNA:Ụkpụrụ nke usoro stem-loop: Ebe ọ bụ na miRNA nile bụ usoro dị mkpirikpi nke ihe dị ka 23 nt, enweghị ike ịchọta PCR kpọmkwem, ya mere a na-eji ngwa usoro nke stem-loop.Usoro nke stem-loop bụ DNA nwere otu eriri nke ihe dịka 50 nt, nke nwere ike ịmepụta nhazi ntutu n'onwe ya.3 'Enwere ike ịmepụta njedebe dị ka usoro na-akwado akụkụ akụkụ miRNA, mgbe ahụ, enwere ike ijikọ miRNA lekwasịrị anya na usoro nkwụsịtụ n'oge ntụgharị ntụgharị, na ngụkọta ogologo nwere ike iru 70bp, nke kwekọrọ na ogologo ngwaahịa agbakwunyere nke qPCR kpebiri.Nhazi primer miRNA tailing.
Nchọpụta akọwapụta nkwalite:
Ebe nchekwa data gbawara n'ịntanetị: mgbawa CottonFGD site na myirịta usoro
Mgbawa mpaghara: Tụtụ aka na iji Blast + na-eme mgbawa mpaghara, Linux na macos nwere ike guzobe nchekwa data mpaghara ozugbo, enwere ike ịme usoro win10 mgbe ịwụnye ubuntu bash.Mepụta nchekwa data mgbawa mpaghara na mgbawa mpaghara;mepee ubuntu bash na win10.
Rịba ama: Owu ugwu na owu agwaetiti dị n'oké osimiri bụ ihe ọkụkụ tetraploid, yabụ nsonaazụ mgbawa na-abụkarị egwuregwu abụọ ma ọ bụ karịa.N'oge gara aga, iji NAU cds dị ka nchekwa data iji mee mgbawa nwere ike ịhụ mkpụrụ ndụ ihe nketa abụọ nwere naanị ọdịiche SNP ole na ole.Ọtụtụ mgbe, mkpụrụ ndụ ihe nketa abụọ ahụ enweghị ike ikewa site na imepụta primer, ya mere a na-ewere ha dị ka otu.Ọ bụrụ na enwere indel doro anya, a na-emepụtakarị primer na indel, ma nke a nwere ike iduga na nhazi nke abụọ nke primer The ike n'efu na-adị elu, na-eduga n'ịbelata arụmọrụ nkwalite, ma nke a bụ ihe a na-apụghị izere ezere.

Nchọpụta nhazi nke abụọ nke mbụ:
Nzọụkwụ:mepee oligo 7 → usoro ntinye ntinye → nso sub-window → chekwaa → chọta primer na template, pịa ctrl+D ka ịtọọ ogologo oge → nyochaa usoro dị iche iche nke abụọ, dị ka ahụ nke onwe ya, heterodimer, ntutu isi, mismatch, wdg. Foto abụọ ikpeazụ na Figure 4 bụ ule nyocha nke primers.N'ihi nke n'ihu primer dị mma, ọ dịghị ihe doro anya dimer na hairpin Ọdịdị, ọ dịghị na-aga n'ihu na-emeju bases, na zuru okè uru nke free ike bụ ihe na-erughị 4.5, mgbe azụ primer na-egosi na-aga n'ihu The 6 bases bụ nkwado, na free ike bụ 8.8;na mgbakwunye, dimer dị njọ karị na-apụta na njedebe 3′, na dimer nke ntọala 4 na-esochi anya pụtara.Ọ bụ ezie na ike efu adịghị elu, 3′ dimer Chl nwere ike imetụta nkwubi okwu nkwalite yana arụmọrụ nkwalite.Na mgbakwunye, ọ dị mkpa ịlele ntutu isi, heterodimers, na mismatches.

Fig3 oligo7 nchọpụta nchọpụta
Nchọpụta arụmọrụ mmụba:
Nkwalite arụmọrụ nke mmeghachi omume PCR na-emetụta nsonaazụ PCR nke ukwuu.Nakwa na qRT-PCR, arụmọrụ mmụba dị mkpa karịsịa maka nsonaazụ ọnụọgụ.Wepu ihe ndị ọzọ, igwe na protocol na nchekwa mmeghachi omume.Ogo nke primers nwekwara mmetụta dị ukwuu na nrụpụta nkwalite nke qRT-PCR.Iji hụ na izi ezi nke nsonaazụ ya, ma ọnụọgụ fluorescence nke ikwu na ọnụọgụ fluorescence zuru oke kwesịrị ịchọpụta nrụpụta nkwalite nke primers.Achọpụtara na arụmọrụ qRT-PCR dị irè dị n'etiti 85% na 115%.Enwere ụzọ abụọ:
1. Usoro mgbagharị ọkọlọtọ:
a.Gwakọta cDNA
b.Dilution gradient
c.qPCR
d.Usoro nkwụghachi azụ Linear iji gbakọọ arụmọrụ mmụba
2. LinRegPCR
LinRegPCR bụ mmemme maka nyocha nke ezigbo oge RT-PCR Data, nke a na-akpọkwa PCR quantitative (qPCR) data dabere na SYBR Green ma ọ bụ kemistri yiri ya.Ihe omume a na-eji data emezigharị na-abụghị nke ndabere, na-eme mgbazi ntọala na nlele ọ bụla iche iche, na-ekpebi windo-nke linearity wee jiri nyocha regression linear dabara n'ahịrị kwụ ọtọ site na data PCR.Site na mkpọda ahịrị a, a na-agbakọ arụmọrụ PCR nke nlele ọ bụla.A na-eji arụmọrụ PCR pụtara n'otu amplicon yana uru Ct kwa nlele iji gbakọọ ntinye uche n'otu n'otu, nke egosipụtara na nkeji fluorescence aka ike.Ntinye data na mmepụta sitere na mpempe akwụkwọ Excel.Naanị sample
Achọrọ ịgwakọta, ọ dịghị gradient
achọrọ usoro:( Were Bole CFX96 dị ka ọmụmaatụ, ọ bụghị ezigbo igwe nwere ABI doro anya)
nnwale:ọ bụ nnwale qPCR ọkọlọtọ.
mmepụta data qPCR:LinRegPCR nwere ike ịmata ụdị abụọ nke faịlụ mmepụta: RDML ma ọ bụ quantification Amplification.N'ezie, ọ bụ uru nchọpụta oge nke nọmba okirikiri na mgbaàmà fluorescence site na igwe, a na-enweta nkwalite ahụ site na nyochaa uru mgbanwe fluorescence nke arụmọrụ nke akụkụ linear.
Nhọrọ data: Na tiori, uru RDML kwesịrị iji ya mee ihe.A na-eme atụmatụ na nsogbu nke kọmputa m bụ na ngwanro enweghị ike ịmata RDML, yabụ enwere m uru mmepụta excel dị ka data mbụ.Ọ na-atụ aro ịrụ a siri ike screening nke data mbụ, dị ka ọdịda nke na-agbakwụnye samples, wdg The isi nwere ike ihichapụ na mmepụta data (n'ezie, ị pụghị ihichapụ ha, LinRegPCR ga-eleghara ihe ndị a na mgbe e mesịrị ogbo).

Fig5qPCR mbupụ data

Fig6 nhọrọ nke atụ ndoro-ndoro ochichi

Ntinye data:Mepee nsonaazụ nkwalite ntozu.xls, → mepee LinRegPCR → faịlụ → gụọ site na excel → họrọ paramita dị ka egosiri na foto 7 → OK → pịa chọpụta baselines

Nzọụkwụ 7 nke ntinye data linRegPCR

Nsonaazụ:Ọ bụrụ na enweghị ikwugharị, ọnweghị nchịkọta achọrọ.Ọ bụrụ na enwere ugboro ugboro, enwere ike ịdezi nchịkọta ahụ na nchịkọta nlele, na aha mkpụrụ ndụ ihe nketa na-abanye na njirimara, mgbe ahụ, a ga-achịkọta otu mkpụrụ ndụ ihe nketa na-akpaghị aka.N'ikpeazụ, pịa faịlụ ahụ, bupụ Excel, wee lelee nsonaazụ ya.A ga-egosipụta arụmọrụ nkwalite yana nsonaazụ R2 nke olulu mmiri ọ bụla.Nke abuo, ọ bụrụ na ị kewaa n'ime otu, a ga-egosipụta arụmọrụ nkwalite agbaziri agbaziri.Gbaa mbọ hụ na arụmọrụ nkwalite nke primer ọ bụla dị n'etiti 85% na 115%.Ọ bụrụ na ọ buru oke ibu ma ọ bụ dị ntakịrị, ọ pụtara na nrụpụta ọrụ nke primer adịghị mma.

Fig 8 Nsonaazụ na mmepụta data

Usoro nnwale:
Ọdịmma RNA chọrọ:
Ịdị ọcha:1.72.0 na-egosi na enwere ike ịnwe isothiocyanate fọdụrụnụ.Nucleic acid A260 / A230 dị ọcha kwesịrị ịdị gburugburu 2. Ọ bụrụ na enwere ike ịmịnye na 230 nm, ọ na-egosi na e nwere ogige ndị dị ka ion phenate.Na mgbakwunye, enwere ike ịchọpụta ya site na 1.5% agarose gel electrophoresis.Tụtụ aka n'ihe nrịbama ahụ, n'ihi na ssRNA enweghị denaturation yana logarithm arọ molekụla enweghị njikọ kwụ ọtọ, na ịdị arọ molekụla enweghị ike igosipụta nke ọma.Ntinye uche: N'echicheọ bụghịIhe na-erughị 100ng / ul, ọ bụrụ na ịta ahụhụ dị oke ala, ịdị ọcha na-adịkarị ala adịghị ogologo

Fig9 RNA gel

Na mgbakwunye, ọ bụrụ na ihe nlele ahụ dị oke ọnụ ahịa yana itinye uche RNA dị elu, a na-atụ aro ka ị wepụta ya mgbe mmịpụta gasịrị, wee tụgharịa RNA na ntinye ikpeazụ nke 100-300ng / ul maka ntụgharị ntụgharị.N'imeusoro ntụgharị ntụgharị, mgbe a na-edegharị mRNA, oligo (dt) primers nke nwere ike jikọta na ọdụ polyA ka a na-eji maka ntụgharị ntụgharị, ebe lncRNA na circRNA na-eji random hexamer (Random 6 mer) primers maka ntụgharị ntụgharị nke mkpokọta RNA Maka miRNA, a na-eji transcription olu-kpọmkwem miRNA mee ihe.Ọtụtụ ụlọ ọrụ ewepụtala ngwa ọdụ ọdụ pụrụ iche ugbu a.Maka usoro stem-loop, usoro ọdụ ọdụ na-adaba adaba, nke dị elu, yana ịchekwa reagent, mana mmetụta nke ịmata ọdịiche nke miRNA nke otu ezinụlọ ekwesịghị ịdị mma ka usoro stem-loop.Ngwa ntụgharị ntụgharị ọ bụla nwere ihe achọrọ maka ntinye nke mkpụrụ ndụ ihe nketa kpọmkwem (stem-loops).Ntụaka dị n'ime ejiri maka miRNA bụ U6.N'ime usoro ntụgharị ntụgharị nke stem-loop, a ga-atụgharị tube nke U6 iche iche, a ga-etinyekwa primers n'ihu na azụ nke U6 ozugbo.Ma circRNA na lncRNA nwere ike iji HKG dịka ntụaka ime.N'imenchọpụta cDNA,
ọ bụrụ na enweghị nsogbu na RNA, cDNA kwesịkwara ịdị mma.Agbanyeghị, ọ bụrụ na a na-achụso izu oke nke nnwale ahụ, ọ kacha mma iji mkpụrụ ndụ nrụtụ aka n'ime (Reference gene, RG) nke nwere ike ịmata ọdịiche gDNA na cds.N'ozuzu, RG bụ mkpụrụ ndụ nchekwa ụlọ., HKG) dị ka e gosiri na foto 10;N'oge ahụ, m na-eme protein nchekwa soybean, ma jiri actin7 nwere introns dị ka ntụaka dị n'ime.Ogo nke mpekere agbakwunyere nke primer a na gDNA bụ 452bp, ma ọ bụrụ na ejiri cDNA dị ka ndebiri, ọ bụ 142bp.Mgbe ahụ, nsonaazụ ule chọpụtara na akụkụ nke cDNA bụ gDNA meruru n'ezie, ma gosipụtakwa na ọ nweghị nsogbu na nsonaazụ ntụgharị ntụgharị, enwere ike iji ya dị ka ndebiri maka PCR.Ọ baghị uru iji cDNA na-agba agarose gel electrophoresis ozugbo, ọ bụkwa eriri mgbasa, nke na-adịghị ekweta.

Fig 10 nchọpụta cDNA

Mkpebi nke ọnọdụ qPCRbụ n'ozuzu enweghị nsogbu dị ka protocol nke kit, tumadi na nzọụkwụ nke tm uru.Ọ bụrụ na ụfọdụ primers na-adịghị mma e mere n'oge primer imewe, na-eme ka a nnukwu ọdịiche dị n'etiti tm uru na theoretical 60 Celsius C, a na-atụ aro na cDNA Mgbe samples na-agwakọta, na-agba ọsọ a gradient PCR na primers, na-agbalị izere ịtọ okpomọkụ na-enweghị ìgwè dị ka TM uru.

Nyocha data

Usoro nhazi fluorescence quantitative PCR bụ nke ọma dabere na 2- ΔΔCT.Ndebiri nhazi data.

Ngwaahịa ndị emetụtara:

Ezigbo oge PCR dị mfe^TM - Taqman

Ezigbo oge PCR dị mfe^TM -SYBR GREEN I

RT Easy I (Master Premix maka eriri cDNA mbụ)

RT Easy II (Master Premix maka njikọ cDNA mbụ maka qPCR)