1. Aqoonta aasaasiga ah (haddii aad rabto inaad aragto qaybta tijaabada, fadlan si toos ah ugu wareeji qaybta labaad)
Dareen-celinta PCR-ga caadiga ah, PCR-da dhabta ah waxay inta badan la socotaa isbeddelka qaddarka badeecada-kordhinta ee wareeg kasta ee falcelinta-weynaynta PCR wakhtiga dhabta ah iyada oo loo marayo beddelka calaamadda fluorescence, oo qiyaas ahaan u falanqeysa qaabka bilowga iyada oo loo marayo xiriirka ka dhexeeya qiimaha ct iyo qalooca caadiga ah.
Xogta gaarka ah ee RT-PCR waagundhig, heerka fluorescenceiyoQiimaha Ct
| gundhigga: | Qiimaha fluorescence ee wareegga 3aad-15aad waa aasaaska (saldhigga), kaas oo ay sababtay khaladka marmar ee cabbirka. |
| Xadka (xadka): | Waxa loola jeedaa xadka ogaanshaha fluorescence ee lagu dejiyay booska ku habboon ee gobolka kobaca jibbaarada ee qalooca cod-weyneysiinta, guud ahaan 10 jeer ka weecida caadiga ah ee salka. |
| Qiimaha CT: | Waa tirada wareegyada PCR marka qiimaha fluorescence ee tuubada falcelinta kasta uu gaaro heerka. Qiimaha Ct wuxuu u dhigma inta uu le'eg yahay qaabka hore. |
Hababka calaamadinta caadiga ah ee RT-PCR:
| habka | faa'iido | nusqaan | baaxadda codsiga |
| SYBR Cagaaran | Codsi ballaadhan, xasaasi ah, raqiis ah oo habboon | Shuruudaha asaasiga ahi aad ayay u sarreeyaan, una nugul baararka aan gaarka ahayn | Waxay ku habboon tahay falanqaynta tirada ee hiddo-sidaha kala duwan ee bartilmaameedka ah, cilmi-baarista muujinta hidda-wadaha, iyo cilmi-baarista xayawaanka iyo dhirta dib-u-soo-celinta. |
| TaqMan | Gaar ahaan wanaagsan iyo soo celinta sare | Qiimuhu waa sarreeyaa wuxuuna ku habboon yahay oo keliya yoolal gaar ah. | Ogaanshaha cudur-sidaha, cilmi-baarista hidda-wadaha iska caabbinta daroogada, qiimeynta waxtarka daawada, ogaanshaha cudurrada hidda-socodka. |
| Ilays molecular | Gaar ahaan sare, fluorescence, asalka hoose | Qiimaha waa sarreeyaa, waxay ku habboon tahay kaliya ujeedo gaar ah, naqshadeynta waa adag tahay, qiimuhuna waa sarreeyaa. | Falanqaynta hidda-sidaha gaarka ah, falanqaynta SNP |
2. Tallaabooyinka tijaabada ah
2.1 Ku saabsan kooxaynta tijaabada ah- Kooxda waa inay jiraan ceelal badan, waana inay jiraan soo noqnoqoshada noolaha.
| ① | Xakamaynta bannaan | Loo isticmaalo in lagu ogaado heerka koritaanka unugga ee tijaabooyinka |
| ② | Xakamaynta xun siRNA (isku xigxiga siRNA aan gaar ahayn) | Muuji sida gaarka ah ee falka RNAi.siRNA waxa laga yaabaa inay keento jawaab celin walaac oo aan gaar ahayn marka la eego 200nM. |
| ③ | Xakamaynta reagent ka gudbinta | Ka saar sunta reagent wareejinta unugyada ama saamaynta muujinta hidda-wadaha la beegsanayo |
| ④ | siRNA lid ku ah hidda-sidaha bartilmaameedka ah | Ku garaac muujinta hidda-wadaha bartilmaameedka ah |
| ⑤ (ikhtiyaar) | siRNA togan | Loo isticmaalo in lagu ciladeeyo nidaamka tijaabada iyo dhibaatooyinka hawlgalka |
| ⑥ (ikhtiyaar) | Xakamaynta fluorescent siRNA | Waxtarka isu-gudbinta unugyada waxaa lagu arki karaa mikroskoob |
2.2 Mabaadi'da naqshadaynta asaasiga ah
| Cabbirka jajabka ee la xoojiyay | Ugu fiican 100-150bp |
| Dhererka Koowaad | 18-25bp |
| nuxurka GC | 30% -70%, gaar ahaan 45% -55% |
| Qiimaha Tm | 58-60 ℃ |
| Isku xigxiga | Iska ilaali T/C si joogto ah;A/G joogto ah |
| 3 isku xigxiga | Ka fogow hodanka GC ama taajirnimada;saldhigga terminal ayaa doorbidaya G ama C;waxaa fiican inaad iska ilaaliso T |
| Dhammaystirnaan | Ka fogow isku xigxiga is kaabaya ee in ka badan 3 saldhig oo ku dhex jira furaha ama inta u dhaxaysa laba aasaasiyaal |
| Gaar ahaan | Adeegso raadinta qaraxa si aad u xaqiijiso gaarka u gaarka ah |
①SiRNA waa nooc-gaar ah, iyo taxanaha noocyada kala duwani way kala duwanaan doonaan.
②SiRNA waxay ku jirtaa budo la qaboojiyey, taas oo si adag loo kaydin karo 2-4 toddobaad heerkulka qolka.
2.3 Qalabka ama reagents u baahan in horay loo sii diyaariyo
| Primer (tixraac gudaha ah) | Oo ay ku jiraan hore iyo gadaal laba |
| Horyaal ( hidde-sideyaasha bartilmaameedka ah) | Oo ay ku jiraan hore iyo gadaal laba |
| Bartilmaameedka Si RNA (3 qaybood) | Guud ahaan, shirkadu waxay soo saari doontaa 3 strips, ka dibna waxay dooran doontaa mid ka mid ah saddexda by RT-PCR |
| Qalabka kala wareejinta | Lipo2000 iwm. |
| Qalabka Soo saarista Degdegga ah ee RNA | Soo saarista RNA ka dib gudbinta |
| Xirmada qoraalka dib-u-celinta degdega ah | loogu talagalay isku-darka cDNA |
| Qalabka Kordhinta PCR | 2×Super SYBR Cagaar qPCR Master Mix |
2.4 Marka la eego arrimaha u baahan in fiiro gaar ah loo yeesho tillaabooyinka tijaabada ah ee gaarka ah:
①siRNA habka gudbinta
1. Dajinta, waxaad dooran kartaa saxan 24-ceed ah, saxan 12-ceel ah ama saxan 6-ceel ah (celceliska fiirsashada RNA ee lagu soo jeediyay ceel kasta oo saxan 24 ah waa qiyaastii 100-300 ng / uL), iyo cufnaanta gudbinta ugu fiican ee unugyada waa ilaa 60% -80% ama wax ka badan.
2. Tallaabooyinka wareejinta iyo shuruudaha gaarka ah ayaa si adag u raacaya tilmaamaha.
3. Gudbinta ka dib, muunado waxaa lagu soo qaadi karaa 24-72 saacadood gudahood si loo ogaado mRNA (RT-PCR) ama ogaanshaha borotiinka 48-96 saacadood gudahood (WB)
② habka soo saarista RNA
1. Kahortagga wasakheynta enzymes dibadda ah.Inta badan waxaa ka mid ah xirashada waji-gashiga iyo galoofyada si adag;iyadoo la isticmaalayo talooyinka pipette ee jeermis dilay iyo tuubooyinka EP;Biyaha loo isticmaalo tijaabada waa inay ahaadaan kuwo ka xor ah RNase.
2. Waxaa lagu talinayaa in la sameeyo laba jeer sida lagu soo jeediyay xirmada soo saarista degdega ah, taas oo runtii hagaajin doonta nadiifnimada iyo dhalidda.
3. dareeraha wasakhda ah waa in aanu taaban tiirka RNA.
③ Tirada RNA
Ka dib marka RNA la soo saaro, waxaa si toos ah loogu qiyaasi karaa Nandrop, iyo akhriska ugu yar wuxuu noqon karaa mid hooseeya sida 10ng / ul.
④ Dib u noqo habka qoraalka
1. Sababtoo ah dareenka sare ee RT-qPCR, ugu yaraan 3 ceel oo isbarbar socda waa in loo sameeyaa muunad kasta si looga hortago in Ct-ga xiga uu aad uga duwanaado ama SD uu aad ugu weynaado falanqaynta tirakoobka.
2. Ha barafaysan oo dhalaali Master isku darka marar badan.
3. Tuubo/dalool kasta waa in lagu beddelaa caarad cusub!Si joogto ah ha u isticmaalin caarada pipette isku mid ah si aad ugu darto muunado!
4. Filimka ku xiran saxanka 96 ka dib marka lagu daro saamiga wuxuu u baahan yahay in lagu simo saxan.Way fiicantahay in la centrifuge ka hor inta aan la saarin mishiinka, si dareeraha derbiga tuubada uu hoos u soo qulqulo oo uu ka saaro xumbo hawo.
⑤ Falanqaynta qalooca caadiga ah
| Ma jiro xilli korriin logarithmic ah | Waxaa suurtogal ah in ay aad u badan tahay qaabka template |
| Qiimo CT ma jiro | Tallaabooyinka khaldan ee lagu ogaanayo calaamadaha fluorescent; hoos u dhigista asaasiga ah ama baaritaanka - daacadnimadeeda waxaa lagu ogaan karaa PAGE electrophoresis; qadar aan ku filnayn template; hoos u dhigista qaababka - ka fogaanshaha soo bandhigida wasakhda iyo qaboojinta soo noqnoqda iyo dhalaalida diyaarinta muunada; |
| Ct>38 | Hufnaanta hoos u dhigista;Alaabta PCR aad bay u dheer tahay;Qaybaha falcelinta kala duwan ayaa hoos u dhacay |
| qalooca dhawaaqa toosan | Baadhitaannada waxa laga yaabaa inay qayb ahaan hoos u dhigaan wareegyada barafaysan ee soo noqnoqda ama soo-gaadhista iftiinka |
| Farqiga u dhexeeya godadka nuqulka ah ayaa si gaar ah u weyn | Xalka falcelintu si buuxda uma dhalaalin ama xalalka falcelinta lama qasin;qubayska kulaylka ee qalabka PCR waxaa ku wasakhoobay walxo fluorescent ah |
2.5 Ku saabsan falanqaynta xogta
Falanqaynta xogta ee qPCR waxaa loo qaybin karaa qiyaas qiyaaseed iyo qiyaas dhammaystiran.Tusaale ahaan, unugyada kooxda daawaynta marka la barbar dhigo unugyada kooxda xakamaynta,
Immisa jeer ayuu mRNA ee hidda-wadaha X isbeddelaa, tani waa qiyaas qiyaas;tiro cayiman oo unugyo ah, mRNA-ga hidda-wadaha X
Immisa nuqul ayaa jira, tani waa qiyaas dhammaystiran.Caadi ahaan waxa aan u isticmaalno shaybaarka inta badan waa habka tirada qaraabada ah.Caadiyan,habka 2-Δctwaxaa inta badan loo isticmaalaa tijaabooyinka , markaa habkan kaliya ayaa halkan lagu soo bandhigi doonaa si faahfaahsan.
Habka 2-Δct: Natiijada la helay ayaa ah farqiga u dhexeeya muujinta hiddaha bartilmaameedka ee kooxda tijaabada ah marka loo eego hiddaha bartilmaameedka ee kooxda kantaroolka.Waxaa loo baahan yahay in waxtarka xoojinta hidda-sidaha bartilmaameedka iyo hidde-sidaha tixraaca ee labadaba ay ku dhow yihiin 100%, iyo weecasho qaraabo ah waa inaysan ka badnayn 5%.
Habka xisaabinta waa sida soo socota:
Kooxda xakamaynta Δct = qiimaha ct ee hidda-wadaha bartilmaameedka ee kooxda kantaroolka - ct qiimaha hiddaha tixraaca gudaha ee kooxda kantaroolka
Kooxda tijaabada ah ee Δct = ct qiimaha hiddaha bartilmaameedka ee kooxda tijaabada ah - ct qiimaha hiddaha tixraaca gudaha ee kooxda tijaabada
ΔΔct = Δct kooxda tijaabada ah - kooxda xakamaynta
Ugu dambayntii, xisaabi faraqa badan ee heerka odhaahda:
Beddel laab=2-ΔΔct (oo u dhiganta shaqada sare waa AWOODDA)
Alaabta la xidhiidha:
Qalabka tooska ah ee gacanta ee RT-qPCR
Waqtiga boostada: Meey-20-2023
