1. Ilimi na asali (idan kuna son ganin sashin gwaji, don Allah canja wurin kai tsaye zuwa kashi na biyu)
A matsayin abin da aka samo asali na PCR na al'ada, PCR na ainihi yana sa ido akan canjin adadin samfuran haɓakawa a cikin kowane sake zagayowar ƙimar haɓakawa na PCR a cikin ainihin lokacin ta hanyar canjin siginar kyalli, da ƙididdige ƙididdige samfurin farawa ta hanyar alaƙa tsakanin ƙimar ct da daidaitaccen lanƙwasa.
Takamaiman bayanan RT-PCR sunetushe, bakin kofakumaCt darajar
| asali: | Ƙimar fluorescence na zagayowar 3rd-15th shine tushe (tushe), wanda ke faruwa ta hanyar kuskuren ma'aunin lokaci-lokaci. |
| Ƙofar (kofa): | Yana nufin iyakar gano haske da aka saita a matsayi da ya dace a cikin yanki mai girman girma na lanƙwan ƙarawa, gabaɗaya sau 10 daidaitaccen karkatacciyar hanyar tushe. |
| Darajar CT: | Yawan zagayowar PCR ne lokacin da ƙimar walƙiya a cikin kowane bututu mai amsawa ya kai bakin kofa. Ƙimar Ct ta bambanta da adadin samfurin farko. |
Hanyoyin sawa na gama gari don RT-PCR:
| hanya | amfani | gazawa | iyakokin aikace-aikace |
| SYBR Green | Wide applicability, m, arha kuma dace | Abubuwan buƙatun farko suna da girma, masu saurin kamuwa da makada marasa takamaiman | Ya dace da ƙididdigar ƙididdiga na nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'in iri) da nau'in nau'ikan kiredit sun bincikar kididdigar, bincike kan bayyanar kwayoyin halitta, da bincike kan dabbobi da tsirrai na sake haduwa da transgenic. |
| TaqMan | Kyakkyawan ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai da babban maimaitawa | Farashin yana da girma kuma kawai ya dace da takamaiman manufa. | Gano ƙwayoyin cuta, bincike kan juriya na ƙwayoyi, ƙididdigar ingancin magunguna, gano cututtukan ƙwayoyin cuta. |
| fitilar kwayoyin halitta | Babban ƙayyadaddun ƙayyadaddun haske, haske mai haske, ƙaramin bango | Farashin yana da girma, yana dacewa kawai don takamaiman dalili, ƙirar yana da wahala, kuma farashin yana da girma. | Specific gene analysis, SNP bincike |
2. Matakan gwaji
2.1 Game da rukunin gwaji- Dole ne a sami rijiyoyi da yawa a cikin rukuni, kuma dole ne a sami maimaitawar halittu.
| ① | Ikon komai | An yi amfani da shi don gano matsayin haɓakar tantanin halitta a cikin gwaje-gwaje |
| ② | SiRNA mara kyau (jerin siRNA mara takamaiman) | Nuna takamaiman aikin RNAi.siRNA na iya haifar da martani mara ƙayyadaddun damuwa a yawan 200nM. |
| ③ | Canja wurin Reagent Control | Banda da guba na reagent transfection zuwa sel ko tasirin bayyanar kwayar halittar da aka yi niyya. |
| ④ | siRNA da manufa | Kaddamar da bayanin kwayar halittar da aka yi niyya |
| ⑤ (na zaɓi) | tabbatacce siRNA | Ana amfani dashi don magance tsarin gwaji da matsalolin aiki |
| ⑥ (na zaɓi) | Mai sarrafa fluorescent siRNA | Ana iya lura da ingancin canjin tantanin halitta tare da na'urar hangen nesa |
2.2 Ka'idoji na ƙirar ƙira
| Girman ɓangarorin da aka haɓaka | Zai fi dacewa a 100-150bp |
| Tsawon Farko | 18-25bp |
| GC abun ciki | 30% -70%, zai fi dacewa 45% -55% |
| Tm darajar | 58-60 ℃ |
| Jeri | Guji T / C ci gaba;A/G ci gaba |
| 3 karshen jerin | Guji GC mai arziki ko AT mai arziki;Tushen tashar ya fi dacewa G ko C;yana da kyau a guji T |
| Kammalawa | Guji jerin abubuwan da suka dace na fiye da tushe 3 a cikin firamare ko tsakanin firamare biyu |
| Musamman | Yi amfani da binciken fashewa don tabbatar da ƙayyadaddun ƙayyadaddun abubuwa |
①SiRNA tana da takamaiman nau'in, kuma jerin nau'ikan nau'ikan nau'ikan daban-daban zasu bambanta.
②An tattara SiRNA a cikin busasshiyar foda, wanda za'a iya adana shi sosai har tsawon makonni 2-4 a zazzabi na ɗaki.
2.3 Kayan aiki ko reagents waɗanda ke buƙatar shirya a gaba
| Alamar farko (maganin ciki) | Ciki har da gaba da baya biyu |
| Abubuwan farko (jinin manufa) | Ciki har da gaba da baya biyu |
| Target Si RNA (tubu 3) | Gabaɗaya, kamfanin zai haɗa sassan 3, sannan zaɓi ɗaya daga cikin ukun ta RT-PCR |
| Canja wurin Kit | Lipo2000 da dai sauransu. |
| Kit ɗin Haɓaka Saurin RNA | Don hakar RNA bayan canzawa |
| Kit ɗin Rubutu Mai Sauri | don haɗin cDNA |
| PCR Amplification Kit | 2 × Super SYBR Green qPCR Master Mix |
2.4 Game da batutuwan da ya kamata a kula da su a cikin takamaiman matakan gwaji:
① siRNA tsarin canzawa
1. Don plating, zaku iya zaɓar farantin rijiyar 24, farantin rijiyar 12 ko farantin rijiyar 6 (matsakaicin adadin RNA da aka gabatar a cikin kowace rijiya na farantin rijiyar 24 shine game da 100-300 ng / uL), kuma mafi kyawun ƙwayar cuta na sel shine har zuwa 60% -80% ko makamancin haka.
2. Matakan canja wuri da ƙayyadaddun buƙatun suna daidai da umarnin.
3. Bayan canzawa, ana iya tattara samfurori a cikin sa'o'i 24-72 don gano mRNA (RT-PCR) ko gano furotin a cikin sa'o'i 48-96 (WB)
② Tsarin hakar RNA
1. Hana gurɓatawa ta ƙwayoyin enzymes masu waje.Ya ƙunshi sanya abin rufe fuska da safar hannu sosai;ta yin amfani da tukwici na pipette da aka haifuwa da bututun EP;Ruwan da aka yi amfani da shi a cikin gwajin dole ne ya zama RNase-Free.
2. Ana ba da shawarar yin sau biyu kamar yadda aka ba da shawara a cikin kayan haɓaka mai sauri, wanda zai inganta da gaske da tsabta da yawan amfanin ƙasa.
3. Dole ne ruwan sharar gida ya taɓa ginshiƙin RNA.
③ Ƙididdigar RNA
Bayan an fitar da RNA, ana iya ƙididdige shi kai tsaye tare da Nandrop, kuma mafi ƙarancin karatun na iya zama ƙasa da 10ng/ul.
④ Juya tsarin rubutun
1. Saboda tsananin hankali na RT-qPCR, aƙalla 3 daidaitattun rijiyoyin ya kamata a yi don kowane samfurin don hana Ct na gaba daga zama daban-daban ko SD ya zama babba don ƙididdigar ƙididdiga.
2. Kada a daskare kuma a narke Jagora akai-akai.
3. Kowane bututu / rami dole ne a maye gurbinsu da sabon tip!Kada ku ci gaba da amfani da tip ɗin pipette iri ɗaya don ƙara samfurori!
4. Fim ɗin da aka haɗe zuwa farantin rijiyar 96 bayan ƙara samfurin yana buƙatar yin laushi tare da farantin karfe.Zai fi kyau a saka shi a kan na'ura kafin a saka shi a kan na'ura, don haka ruwan da ke kan bangon bututu zai iya gudana kuma ya cire kumfa na iska.
⑤Bincike na yau da kullun
| Babu lokacin haɓaka logarithmic | Yiwuwar babban taro na samfuri |
| Babu darajar CT | Matakan da ba daidai ba don gano siginar kyalli; lalatawar abubuwan farko ko bincike - ana iya gano amincin sa ta PAGE electrophoresis; rashin isasshen adadin samfuri; lalata samfura - guje wa gabatarwar datti da maimaita daskarewa da narke a cikin shirye-shiryen samfurin; |
| Ct>38 | Ƙarƙashin haɓakar haɓakawa;Samfurin PCR ya yi tsayi da yawa;daban-daban dauki aka gyara suna ƙasƙanta |
| Lanƙwan ƙara girman layi | Ana iya lalata bincike a wani ɗan lokaci ta hanyar maimaita daskarewar hawan keke ko tsawan lokaci ga haske. |
| Bambanci a cikin ramukan kwafi yana da girma musamman | Maganin amsawa ba a narkar da shi gaba daya ko kuma ba a gauraya maganin ba;wanka mai zafi na kayan aikin PCR yana gurɓata da abubuwa masu kyalli |
2.5 Game da nazarin bayanai
Ana iya raba nazarin bayanan qPCR zuwa ƙididdige dangi da cikakken ƙididdigewa.Misali, sel a cikin rukunin jiyya idan aka kwatanta da sel a cikin rukunin kulawa,
Sau nawa mRNA na kwayar halittar X ke canzawa, wannan ƙididdigewa ne;a cikin takamaiman adadin sel, mRNA na kwayar halittar X
Kwafi nawa ne, wannan cikakkiyar ƙididdigewa ce.Yawancin abin da muke amfani da su a cikin dakin gwaje-gwaje shine hanyar ƙididdigewa dangi.Yawancin lokaci,Hanyar 2-ΔΔctana amfani dashi mafi yawa a cikin gwaje-gwaje , don haka kawai wannan hanyar za a gabatar da dalla-dalla a nan.
Hanyar 2-ΔΔct: Sakamakon da aka samu shine bambanci a cikin maganganun da aka yi amfani da shi a cikin ƙungiyar gwaji dangane da kwayar cutar da ke cikin ƙungiyar kulawa.Ana buƙatar cewa ingancin ingancin kayan kwayar halitta da kwayoyin halittar ciki suna kusa da 100%, kuma karkatar da ƙwarewa kada su wuce 5%.
Hanyar lissafin ita ce kamar haka:
Ƙungiyar kula da Δct = ct darajar asalin manufa a cikin ƙungiyar kulawa - ƙimar ct na ƙwayar tunani na ciki a cikin ƙungiyar kulawa
Ƙungiya na gwaji Δct = ct darajar maƙasudin kwayar halitta a cikin ƙungiyar gwaji - ƙimar ct na ƙirar bincike na ciki a cikin ƙungiyar gwaji
ΔΔct = Δct ƙungiyar gwaji-Δct ƙungiyar kulawa
A ƙarshe, ƙididdige bambance-bambance masu yawa a matakin magana:
Canja Ninka = 2-ΔΔct (daidai da aikin Excel shine WUTA)
Samfura masu alaƙa:
Lokacin aikawa: Mayu-20-2023
