1. Chidziwitso choyambirira (ngati mukufuna kuwona gawo loyesera, chonde tumizani ku gawo lachiwiri)
Monga zotumphukira za PCR wamba, Nthawi Yeniyeni PCR makamaka imayang'anira kusintha kwa kuchuluka kwa mankhwala okulitsa mumkombero uliwonse wa PCR amplification reaction mu nthawi yeniyeni kudzera pakusintha kwa siginecha ya fluorescence, ndikuwunika mochulukira template yoyambira kudzera paubwenzi pakati pa mtengo wa ct ndi pamapindikira okhazikika.
Zambiri za RT-PCR ndizoyambira, mphamvu ya fluorescencendiMtengo wapatali wa magawo Ct.
| zoyambira: | Mtengo wa fluorescence wa 3rd-15th cycle ndizoyambira (zoyambira), zomwe zimayambitsidwa ndi zolakwika zanthawi zina za kuyeza. |
| Chiyambi (chiyambi): | Imatanthawuza malire a kuzindikira kwa fluorescence omwe amaikidwa pamalo oyenera m'chigawo cha kukula kwa curve ya amplification, nthawi zambiri ka 10 kupatuka koyambira. |
| Mtengo wa CT: | Ndi chiwerengero cha ma PCR pamene mtengo wa fluorescence mu chubu chilichonse umafika pakhomo. Mtengo wa Ct ndi wosiyana kwambiri ndi kuchuluka kwa template yoyambira. |
Njira zodziwika bwino za RT-PCR:
| njira | mwayi | chopereŵera | kuchuluka kwa ntchito |
| SYBR GreenⅠ | Wide applicability, tcheru, yotsika mtengo komanso yabwino | Zofunikira zoyambira ndizokwera, zomwe zimatengera magulu omwe siatchulidwe | Ndikoyenera kuwunika kuchuluka kwa majini osiyanasiyana omwe amatsata, kafukufuku wama jini, ndi kafukufuku wa nyama ndi zomera zomwe zimasinthanso. |
| TaqMan | Kukhazikika kwabwino komanso kubwereza kwakukulu | Mtengo wake ndi wapamwamba komanso woyenera pa zolinga zenizeni. | Kuzindikira kwa tizilombo toyambitsa matenda, kufufuza kwa majini osagwirizana ndi mankhwala, kuwunika momwe mankhwala amagwirira ntchito, kuzindikira matenda amtundu. |
| ma molekyulu | High specificity, fluorescence, otsika maziko | Mtengo wake ndi wokwera, umangoyenera cholinga chapadera, mapangidwe ake ndi ovuta, ndipo mtengo wake ndi wapamwamba. | Kusanthula kwa majini enieni, kusanthula kwa SNP |
2. Njira zoyesera
2.1 Za gulu loyesera- payenera kukhala zitsime zingapo pagulu, ndipo payenera kukhala kubwereza kwachilengedwe.
| ① | Kuwongolera popanda kanthu | Amagwiritsidwa ntchito pozindikira kukula kwa ma cell poyeserera |
| ② | Negative control siRNA (kutsatizana kosagwirizana ndi siRNA) | Sonyezani tsatanetsatane wa zochita za RNAi.siRNA ikhoza kuyambitsa kuyankha kwapang'onopang'ono kosagwirizana ndi 200nM. |
| ③ | Transfection Reagent Control | Kupatula kawopsedwe wa transfection reagent ku maselo kapena zotsatira za jini chandamale |
| ④ | siRNA motsutsana ndi jini ya chandamale | Gwirani pansi mawu a jini yomwe mukufuna |
| ⑤ (ngati mukufuna) | zabwino siRNA | Amagwiritsidwa ntchito kuthetsa mavuto oyesera ndi ntchito |
| ⑥ (ngati mukufuna) | Fluorescent control siRNA | Kuchita bwino kwa kufalikira kwa maselo kumatha kuwonedwa ndi maikulosikopu |
2.2 Mfundo zoyambira kupanga
| Kukula kwachidutswa chokulitsa | Makamaka pa 100-150bp |
| Utali Woyambira | 18-25bp |
| Zithunzi za GC | 30% -70%, makamaka 45% -55% |
| Mtengo wa Tm | 58-60 ℃ |
| Kutsatizana | Pewani T / C mosalekeza;A/G mosalekeza |
| 3 mapeto atsatizana | Pewani GC wolemera kapena AT wolemera;ma terminal base makamaka G kapena C;ndibwino kupewa T |
| Kukwanirana | Pewani kutsatizana kowonjezera kopitilira 3 zoyambira mkati mwa zoyambira kapena pakati pa zoyambira ziwiri |
| Mwatsatanetsatane | Gwiritsani ntchito kusaka kophulika kuti mutsimikizire zoyambira |
①SiRNA imatengera mitundu, ndipo kutsata kwa mitundu yosiyanasiyana kumakhala kosiyana.
②SiRNA imayikidwa mu ufa wowuma, womwe ukhoza kusungidwa bwino kwa masabata a 2-4 kutentha.
2.3 Zida kapena ma reagents omwe akuyenera kukonzekera pasadakhale
| Primer (zofotokozera zamkati) | Kuphatikizapo kutsogolo ndi kumbuyo ziwiri |
| Zoyambira (majini omwe mukufuna) | Kuphatikizapo kutsogolo ndi kumbuyo ziwiri |
| Target Si RNA (mizere 3) | Nthawi zambiri, kampaniyo ipanga mizere itatu, kenako ndikusankha imodzi mwa zitatuzo ndi RT-PCR |
| Transfection Kit | Lipo2000 etc. |
| RNA Rapid Extraction Kit | Kwa RNA m'zigawo pambuyo kupatsira |
| Rapid Reverse Transcription Kit | kwa cDNA synthesis |
| PCR Amplification Kit | 2 × Super SYBR Green qPCR Master Mix |
2.4 Ponena za zovuta zomwe zikuyenera kutsatiridwa ndi masitepe apadera oyesera:
①SiRNA transfection process
1. Pakuyika, mutha kusankha 24-chitsime mbale, 12-chitsime mbale kapena 6-chitsime mbale (avareji RNA ndende akufuna mu chitsime chilichonse cha 24-chitsime mbale ndi za 100-300 ng/uL), ndipo mulingo woyenera kwambiri kusamvana kachulukidwe maselo ndi mpaka 60 % -80% kapena choncho.
2. Masitepe opatsirana ndi zofunikira zenizeni ndizogwirizana ndi malangizo.
3. Pambuyo pa kupatsirana, zitsanzo zikhoza kusonkhanitsidwa mkati mwa maola 24-72 kuti mudziwe mRNA (RT-PCR) kapena kufufuza mapuloteni mkati mwa maola 48-96 (WB)
② Njira yochotsera RNA
1. Pewani kuipitsidwa ndi michere yakunja.Zimaphatikizapo kuvala masks ndi magolovesi mosamalitsa;kugwiritsa ntchito nsonga za pipette zosawilitsidwa ndi machubu a EP;madzi ogwiritsidwa ntchito poyesera ayenera kukhala RNase-Free.
2. Ndibwino kuti tichite kawiri monga momwe tafotokozera muzitsulo zowonongeka mwamsanga, zomwe zidzasintha kwambiri chiyero ndi zokolola.
3. The zinyalala zamadzimadzi sayenera kukhudza RNA ndime.
③ kuchuluka kwa RNA
Pambuyo pa kuchotsedwa kwa RNA, ikhoza kuwerengedwa mwachindunji ndi Nanodrop , ndipo kuwerenga kochepa kungakhale kochepa ngati 10ng / ul.
④Njira yosinthira yomasulira
1. Chifukwa cha kukhudzidwa kwakukulu kwa RT-qPCR, osachepera 3 zitsime zofanana ziyenera kupangidwa pa chitsanzo chilichonse kuti ateteze Ct yotsatira kuti ikhale yosiyana kwambiri kapena SD kuti ikhale yaikulu kwambiri kuti ifufuze mawerengero.
2. Osazizira ndi kusungunula Master mix mobwerezabwereza.
3. Chubu/bowo lililonse liyenera kusinthidwa ndi nsonga yatsopano!Musagwiritse ntchito nsonga ya pipette kuti muwonjezere zitsanzo!
4. Firimuyi yophatikizidwa ndi mbale ya 96-chitsime pambuyo powonjezera chitsanzocho chiyenera kukonzedwa ndi mbale.Ndibwino kuti muyike centrifuge musanayike pamakina, kuti madzi omwe ali pakhoma la chubu azitha kutsika ndikuchotsa thovu la mpweya.
⑤Kusanthula kozungulira kofala
| Palibe nthawi ya kukula kwa logarithmic | Mwina mkulu kuchuluka kwa template |
| Palibe mtengo wa CT | Masitepe olakwika ozindikira ma siginecha a fulorosenti; kuwonongeka kwa zoyambira kapena zofufuza - kukhulupirika kwake kumatha kudziwika ndi PAGE electrophoresis; kuchuluka kosakwanira kwa template; kuwonongeka kwa ma templates - kupeŵa kuyambitsa zonyansa ndi kuzizira mobwerezabwereza ndi kusungunuka pokonzekera zitsanzo; |
| Mt>38 | Low amplification mphamvu;Pulogalamu ya PCR ndi yayitali kwambiri;zosiyanasiyana anachita zigawo zikuluzikulu ndi oipa |
| Linear amplification curve | Ma probe amatha kuonongeka pang'ono chifukwa cha kuzizira kobwerezabwereza kapena kuyanika kwa nthawi yayitali |
| Kusiyana kwa mabowo obwereza kumakhala kwakukulu kwambiri | Njira yothetsera siisungunuka kwathunthu kapena yankho silinasakanizidwe;kusamba kotentha kwa chipangizo cha PCR kumaipitsidwa ndi zinthu za fulorosenti |
2.5 Zokhudza kusanthula deta
Kusanthula kwa data kwa qPCR kumatha kugawidwa mu kuchuluka kwachibale ndi kuchuluka kwathunthu.Mwachitsanzo, ma cell omwe ali mgulu lachipatala poyerekeza ndi ma cell omwe ali mugulu lolamulira,
Ndi kangati mRNA wa X jini imasintha, uku ndi kuchulukitsidwa kwapang'onopang'ono;m'maselo angapo, mRNA wa X jini
Ndi makope angati omwe alipo, uku ndikuwerengera kwathunthu.Nthawi zambiri zomwe timagwiritsa ntchito kwambiri mu labotale ndi njira yowerengera.Nthawi zambiri,njira ya 2-ΔΔctamagwiritsidwa ntchito kwambiri muzoyesera , kotero njira iyi yokha idzafotokozedwa mwatsatanetsatane apa.
2-ΔΔct njira: Chotsatira chomwe chinapezedwa ndi kusiyana kwa kufotokozera kwa jini yowunikira mu gulu loyesera lokhudzana ndi jini lolunjika mu gulu lolamulira.Ndikofunikira kuti kukulitsa kwamphamvu kwa jini yomwe mukufuna komanso jini yolozera mkati ili pafupi ndi 100%, ndipo kupatuka kwapagulu sikuyenera kupitirira 5%.
Njira yowerengera ili motere:
Δct control gulu = ct mtengo wa chandamale cha jini mu gulu lolamulira - ct mtengo wa jini lothandizira mkati mu gulu lolamulira
Δct experimental gulu = ct mtengo wa jini yowunikira mu gulu loyesera - ct mtengo wa jini lothandizira mkati mu gulu loyesera
Δct = Δct gulu loyesera-Δct gulu lolamulira
Pomaliza, werengani kuchuluka kwa kusiyana kwa mawu:
Sinthani Fold = 2-ΔΔct (yogwirizana ndi ntchito ya Excel ndi MPHAMVU)
Zogwirizana nazo:
Nthawi yotumiza: May-20-2023
