1. Ruzivo rwekutanga (kana iwe uchida kuona chikamu chekuyedza, ndapota endesa zvakananga kune chikamu chechipiri)
Sekuita kwakabva kune kwakajairika PCR, Nguva chaiyo PCR inonyanya kutarisa shanduko yehuwandu hwechigadzirwa chekusimudzira mune yega yega kutenderera kwePCR amplification reaction munguva chaiyo kuburikidza nekushandurwa kweiyo fluorescence chiratidzo, uye nehuwandu hunoongorora yekutanga template kuburikidza nehukama pakati peiyo ct kukosha uye yakajairwa curve.
Iyo chaiyo data yeRT-PCR ndeyebaseline, fluorescence chikumbaridzouyeCt kukosha.
| yekutanga: | Kukosha kwefluorescence ye 3rd-15th cycle ndiyo yekutanga (baseline), iyo inokonzerwa nekamwe kukanganisa kwechiyero. |
| Chikumbaridzo (chikumbaridzo): | Inoreva iyo fluorescence yekuona muganho wakaiswa panzvimbo yakakodzera munzvimbo yekukura kweiyo amplification curve, kazhinji ka10 kutsauka kwakajairwa kwekutanga. |
| CT kukosha: | Ndiyo nhamba yePCR cycles apo kukosha kwefluorescence mune imwe neimwe yekuita tube inosvika pachikumbaridzo. Iko Ct kukosha inopesana nechiyero chekutanga template. |
Nzira dzakajairika dzekunyora dzeRT-PCR:
| nzira | advantage | kushomeka | chiyero chekushandisa |
| SYBR GreenⅠ | Wide applicability, inonzwisisika, yakachipa uye yakanakira | Zvido zvekutanga zvakakwirira, zvinotarisana nemabhendi asina-chaiyo | Yakakodzera kuongororwa kwehuwandu hweakasiyana-siyana anotarirwa majini, kutsvagisa nezve gene expression, uye tsvagiridzo yezve transgenic recombinant mhuka nemiti. |
| TaqMan | Yakanaka chaiyo uye yakakwirira kudzokorora | Mutengo wacho wakakwirira uye wakakodzera chete kune zvinangwa zvakananga. | Pathogen kuonekwa, zvinodhaka kuramba gene tsvakurudzo, zvinodhaka efficacy assessment, kuongororwa kwemajini zvirwere. |
| molecular beacon | High chaiyo, fluorescence, yakaderera kumashure | Mutengo wacho wakakwirira, unokodzera chete chinangwa chaicho, dhizaini yakaoma, uye mutengo wakakwirira. | Yakananga gene kuongororwa, SNP kuongororwa |
2. Kuedza matanho
2.1 Nezvekuunganidzwa kwekuedza- panofanirwa kunge paine matsime akawanda muboka, uye panofanirwa kuve nekudzokororwa kwebiological.
| ① | Kudzora kusina chinhu | Inoshandiswa kuona chimiro chekukura kwesero muzviedzo |
| ② | Negative control siRNA (isina-specific siRNA sequence) | Ratidza iyo chaiyo yeRNAi chiito.siRNA inogona kukonzera kusiri-chaiyo kushushikana kupindurwa pakusangana kwe200nM. |
| ③ | Transfection Reagent Control | Usasanganise huturu hweiyo transfection reagent kumaseru kana mhedzisiro pakutaura kweiyo gene gene |
| ④ | siRNA inopesana nechinangwa chejeni | Ronza pasi kutaura kwejini rinotarirwa |
| ⑤ (kusarudza) | positive siRNA | Inoshandiswa kugadzirisa kuyedza system uye matambudziko ekushanda |
| ⑥ (sarudzo) | Fluorescent control siRNA | Kushanda kwesero transfection kunogona kuonekwa ne microscope |
2.2 Nheyo dzekutanga dhizaini
| Amplified fragment size | Zviri nani pa100-150bp |
| Primer Length | 18-25bp |
| GC zvemukati | 30% -70%, zvichida 45% -55% |
| Tm value | 58-60 ℃ |
| Zvinoteverana | Dzivisa T / C inoenderera;A/G inoenderera |
| 3 kupera kutevedzana | Dzivisa GC mupfumi kana AT akapfuma;iyo terminal base inodiwa G kana C;zvakanaka kudzivisa T |
| Kuwirirana | Dzivisa kutevedzana kwekutevedzana kweanopfuura matatu mabhesi mukati mekutanga kana pakati pekutanga maviri |
| Zvakananga | Shandisa blast search kuti usimbise primer chaiyo |
①SiRNA imhando-chaiyo, uye kutevedzana kwemarudzi akasiyana kuchave kwakasiyana.
②SiRNA inoputirwa muhupfu yakaomeswa nechando, inogona kuchengetwa yakatsiga kwemavhiki maviri-4 patembiricha yekamuri.
2.3 Zvishandiso kana reagents zvinoda kugadzirirwa pachine nguva
| Primer (referensi yemukati) | Kusanganisira mberi uye kumashure maviri |
| Primers (targe gene) | Kusanganisira mberi uye kumashure maviri |
| Target Si RNA (3 mitsetse) | Kazhinji, kambani ichagadzira 3 mitsetse, uye wozosarudza imwe yeatatu neRT-PCR |
| Transfection Kit | Lipo2000 etc. |
| RNA Rapid Extraction Kit | Yekubvisa RNA mushure mekutapurirana |
| Rapid Reverse Transcription Kit | ye cDNA synthesis |
| PCR Amplification Kit | 2 × Super SYBR Green qPCR Master Mix |
2.4 Nezve nyaya dzinoda kutariswa mumatanho chaiwo ekuedza:
①siRNA transfection process
1. Pakuputira, unogona kusarudza 24-tsime ndiro, 12-tsime ndiro kana 6-tsime ndiro (avhareji RNA concentration inorongwa mutsime rimwe nerimwe re24-tsime ndiro inenge 100-300 ng/uL), uye optimal transfection density yemasero anosvika 60 % -80% kana zvakadaro.
2. Matanho ekutapurirana uye zvinodiwa zvakananga zvakanyatsoenderana nemirairo.
3. Mushure mekutapurirwa, sampuli dzinogona kuunganidzwa mukati meawa 24-72 yekuona mRNA (RT-PCR) kana kuonekwa kweprotein mukati maawa 48-96 (WB)
② RNA kuburitsa maitiro
1. Kudzivirira kusvibiswa nemaenzayimu ekunze.Inonyanya kusanganisira kupfeka masiki uye magurovhosi zvakasimba;kushandisa sterilized pipette mazano uye EP tubes;mvura inoshandiswa mukuedza inofanira kunge iri RNase-Free.
2. Inokurudzirwa kuita kaviri sezvinoratidzwa muchikwata chekutsvaga chinokurumidza, icho chichanyatso kuvandudza kuchena uye goho.
3. Mushonga wemarara haufanire kubata RNA column.
③ RNA quantification
Mushure mokunge RNA yabviswa, inogona kuverengwa zvakananga neNanodrop, uye zvishoma zvishoma kuverenga zvinogona kunge zvakaderera se10ng / ul.
④Reverse transcription process
1. Nekuda kwekunzwa kwakanyanya kweRT-qPCR, zvishoma 3 matsime akaenzana anofanirwa kuitwa kune imwe neimwe sample kudzivirira iyo inotevera Ct kubva kune yakanyanyisa kusiyana kana SD kuti ive yakakura kwazvo pakuongorora nhamba.
2. Usaomesa nechando uye kunyungudusa Master mix kakawanda.
3. Imwe neimwe chubhu / gomba rinofanira kutsiviwa netipi nyowani!Usarambe uchishandisa iyo imwechete pipette tipi yekuwedzera samples!
4. Iyo firimu yakasungirirwa kune 96-tsime ndiro mushure mekuwedzera sampuli inoda kugadziriswa neplate.Zvakanakisisa kuisa centrifuge usati waiisa pamushini, kuitira kuti mvura iri pamadziro echubhu inogona kuyerera ichidzika uye kubvisa mabhubhu emhepo.
⑤Common curve analysis
| Hapana nguva yekukura kwe logarithmic | Zvingangove zvakakwirira zvema template |
| Hapana CT kukosha | Matanho asina kururama ekuona masaini efluorescent; kuderedzwa kwekutanga kana probes - kuvimbika kwayo kunogona kuonekwa nePEJI electrophoresis; huwandu husina kukwana hwetemplate; kushatiswa kwematemplate - kudzivirira kuiswa kwetsvina uye kudzokororwa nechando uye kunyunguduka mukugadzirira kwemuenzaniso; |
| Ct>38 | Low amplification kunyatsoshanda;PCR chigadzirwa chakareba kwazvo;zvakasiyana-siyana zvekuita zvikamu zvakasvibiswa |
| Linear amplification curve | Probes inogona kuderedzwa zvishoma nekudzokororwa kwechando-kunyunguduka kana kuratidzwa kwenguva refu kuchiedza. |
| Musiyano wemakomba akapetwa wakakura zvakanyanya | Mhinduro yekugadzirisa haina kunyungudutswa zvachose kana mhinduro yekugadzirisa haina kusanganiswa;iyo inopisa yekugezera yePCR chiridzwa yakasvibiswa nezvinhu zvefluorescent |
2.5 Pamusoro pekuongorora data
Iyo data yekuongorora yeqPCR inogona kukamurwa kuita hukama hwehuwandu uye yakakwana quantification.Semuenzaniso, masero ari muboka rekurapa achienzaniswa nemasero ari muboka rekutonga,
Kangani iyo mRNA yeX gene inoshanduka, uku ndiko kuenzana kwehuwandu;mune imwe nhamba yemasero, iyo mRNA yeX gene
Mangani makopi aripo, uku ndiko kukwana kwehuwandu.Kazhinji chatinoshandisa zvakanyanya murabhoritari ndiyo nzira yehuwandu.Kazhinji,iyo 2-ΔΔct nzirandiyo inoshandiswa zvakanyanya mukuedzwa, saka nzira iyi chete ndiyo ichaunzwa zvakadzama pano.
2-ΔΔct nzira: Chigumisiro chakawanikwa ndicho mutsauko mukutaura kwejini rinotarirwa muboka rekuedza maererano nejini rinotarirwa muboka rekutonga.Zvinotarisirwa kuti kukwidziridzwa kwemajini uye yemukati referensi gene iri padyo ne100%, uye kutsauka kwacho hakufanire kudarika 5%.
Nzira yekuverenga ndeyotevera:
Δct control boka = ct kukosha kwechinangwa gene muboka rekutonga - ct kukosha kwemukati referensi gene muboka rekutonga
Δct yeboka rekuedza = ct kukosha kwejini rinotarirwa muboka rekuedza - ct kukosha kwemukati rekutsvaga gene muboka rekuedza.
Δct = Δct yeboka rekuedza-Δct control boka
Pakupedzisira, verenga kuwanda kwemutsauko padanho rekutaura:
Shandura Fold = 2-ΔΔct (inoenderana neexcel basa iSIMBA)
Zvigadzirwa zvinoenderana:
Nguva yekutumira: May-20-2023
