RNA (dsRNA) labajibbaaran ee ELISA KIT

Xirmadani waa Enzyme-Linked Immunosorbent Assay (ELISA) oo ku xidhan nidaamka biotin-Streptavidin, ee cabbiraadda tirada ee dsRNA oo dhererkeedu ka sarreeyo 60 lammaane sal (bp), iyadoon loo eegin sida ay isugu xigaan.Saxanka waxa hore loogu dahaadhay anti-dsRNA antibody.dsRNA ee ku jirta muunada ayaa lagu daraa oo lagu xidhaa unugyada difaaca jirka ee lagu dahaadhay ceelasha.Ka dibna biotinylated anti-dsRNA antibody ayaa lagu daraa oo ku xidhaa dsRNA muunada.Ka dib marka la dhaqo, HRP-Streptavidin waa lagu daraa oo waxay ku xidhaa antibody-ka Biotinylated anti-dsRNA.Ka dib marka la isku xidho HRP-Streptavidin waa la dhaqay.Kadibna xalka TMB substrate ayaa lagu daraa oo ay kicisay HRP si ay u soo saarto badeeco midab buluug ah oo isu beddeshay huruud ka dib marka lagu daro xal joojinta acidic.Cufnaanta jaalaha waxay u dhigantaa qiyaasta bartilmaameedka ee dsRNA ee lagu qabtay saxanka.Nuugista waxaa lagu qiyaasaa 450 nm.

Codsiga

Qalabkan waxaa loogu talagalay cabbiraadda tirada ee dsRNA haraaga.

Qaybaha Kit

Kaydinta iyo Xasiloonida

1. Qalabka aan la isticmaalin: Qalabka oo dhan waxaa lagu kaydin karaa 2 ~ 8 ℃ nolosha shelf.Iftiin xooggan waa in laga fogaadaa xasilloonida kaydinta.

2. Qalabka la isticmaalay: Marka microplate-ka la furo, fadlan ku dabool ceelasha aan la isticmaalin saxan-xeeraha oo ku soo celi boorsada bireed ee ay ku jiraan baakidhka qallajiya, sib-ka xidh kiishka bireedyada oo ku soo celi 2 ~ 8 ℃ sida ugu dhakhsaha badan ee suurtogalka ah isticmaalka ka dib.Reageniyeyaasha kale waa in lagu soo celiyaa 2 ~ 8 ℃ sida ugu dhakhsaha badan ee suurtogalka ah ka dib isticmaalka.

Alaabta Loo Baahan Yahay Balse Aan La Bixin

1. Akhristaha Microplate oo leh 450 ± 10nm filter (ka wanaagsan haddii lagu ogaan karo 450 iyo 650 nm mawjada).

2. Microplate ruxul.

3. Talooyin aan RNase-la'aan ahayn iyo tuubooyinka centrifuge.

Jaantuska socodka hawlgalka

Inta aanad bilaabin

1. Keen dhammaan qaybaha qalabka iyo muunadaha heerkulka qolka (18-25 ℃) kahor isticmaalka.Haddii xirmada aan la isticmaali doonin hal mar, fadlan kaliya soo saar xayndaabyada iyo reagents tijaabada hadda, oo ka tag xariijinta iyo reagents-ka soo hadhay xaalad loo baahan yahay.

2. Bakhaarada dhaqida: ku qas 40mL oo ah 20× 760mL oo biyo la miiray ama la dhaqay si aad u diyaariso 800mL oo ah 1× wax dhaqida.

3. Halbeeg: si kooban u miiqji ama sentrifuge xalka kaydka ka hor inta aan la isticmaalin.Xaddiga afar heerar oo la bixiyay waa 5ng/μL.Heerarka UTP iyo pUTP dsRNA, fadlan ku mili xalka saamiga 1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL oo leh STE kaydiye si aad u sawirto qalooca caadiga ah.Heerarka N1-Me-pUTP dsRNA, fadlan ku milmi xalka kaydka 2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL oo leh STE kaydiye si aad u sawirto qalooca caadiga ah.Halbeegga 5-OMe-UTP dsRNA, fadlan ku mili xalka saamiga 4,2,1,0.5, 0.25,0.125,0.0625, 0pg/μL oo leh STE kaydiye si aad u sawirto qalooca caadiga ah.Waxaan ku talinaynaa in heerarka lagu qasi karo sida jaantusyada soo socda:

4. Biotinylated antibody detection antibody iyo HRP-streptavidin xal u shaqaynaysa: si kooban u miiqji ama sentrifuge xalka kaydka ka hor inta aan la isticmaalin.Ku qas iyaga oo u fiirsashada shaqada adigoo isticmaalaya bakhtiiye.

5. TMB substate: hami qiyaasta loo baahan yahay ee xalka adigoo isticmaalaya talooyin jeermis dilay oo ha ku daadin xalka haraaga mar labaad.Dawlad-hoosaadyada TMB waxay xasaasi u tahay iftiinka, ha u soo bandhigin substrate-ka TMB iftiinka muddo dheer.

Isticmaalka borotokoolka

1. Go'aami tirada xariijinta ee loo baahan yahay baaritaanka.Geli xariijimaha fiimyada si loo isticmaalo.Xarigga saxan ee haray ee aan loo isticmaalin baaritaankan waa in lagu soo celiyaa bacda iyada oo la qalajiyey.Si adag ugu xidh bacda kaydinta qaboojiyaha.

2. Ku dar 100μL mid kasta oo ka mid ah qasiyadaha heerka caadiga ah, maran iyo muunado ceelasha ku habboon.Ku dabool xiraha saxanka.Ku duri 1 saac heerkulka qolka adigoo gariiraya 500rpm.Tijaabooyinku waa in lagu qasi karaa STE kaydiye si ay ugu fiirsadaan si sax ah loo qiimeeyo.

3. Talaabada dhaq: Ka dami xalka oo ku maydh 250μL meel kasta oo wax lagu dhaqo oo u daa inuu istaago 30s.Si buuxda u tuur bakhaarka dhaqida adiga oo saxanka dul saaraya warqad nuugista.Gebi ahaanba maydh 4 jeer.

4. Ku dar 100μL oo ah antibody ogaanshaha biotinylated ee ceel kasta.Ku dabool xiraha saxanka.Ku duri 1 saac heerkulka qolka adigoo gariiraya 500rpm.

5. Ku celi talaabada dhaqida.

6. Ku dar 100μL ee HRP-streptavidin xal shaqaynaysa ceel kasta.Ku dabool xiraha saxanka.Ku dhaji 30min heerkulka qolka adigoo gariiraya 500rpm.

7. Ku celi talaabada dhaqida mar kale.

8. Ceel walba ku dar 100μL oo substrate TMB ah.Ku dabool xiraha saxanka.Ku duri 30 daqiiqo at RT Ka ilaali iftiinka.Dareeraha wuxuu isu rogi doonaa buluug marka lagu daro xal substrate ah.

9. Ceel walba ku dar 50μL oo xal joogsi ah.Dareeraha wuxuu isu rogi doonaa huruud marka lagu daro xalka joogsiga.Dabadeed orod akhriyaha microplate oo samee cabbirka 450nm isla markaaba.

Xisaabinta Natiijooyinka

1. Isku celcelis akhrinta nuqulka ah ee halbeeg kasta, kantarool, iyo muunado oo ka jar celceliska cufnaanta caadiga ah ee eber.Ku dhis qalooca caadiga ah oo leh nuugista dhidibka toosan (Y) iyo fiirsashada dsRNA ee dhinaca toosan (X)

2. Waxaa lagu talinayaa in lagu sameeyo xisaabinta iyada oo la adeegsanayo software-kumbuyuutar ku salaysan qalooca sida khabiirka qalooca 1.3 ama ELISA Calc oo ah 5 ama 4 parameter oo aan toos ahayn.

Waxqabadka

1. Xasaasiyadda:

xadka hoose ee ogaanshaha: ≤ 0.001pg/μL (UTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (loogu talagalay 5-OMe-UTP-dsRNA).

Xadka hoose ee tirada:0.0156 pg/μL (UTP-, pUTP-dsRNA),0.0312 pg/μL (loogu talagalay N1-Me-pUTP-dsRNA),0.0625 pg/μL (loogu talagalay 5-OMe-UTP-dsRNA).

2. Saxnaanta: CV of Intra-Assay ≤10%, CV ee Inter-Assay ≤10%

3. Soo kabashada: 80% ~ 120%

4.Linearity: 0.0156-0.5pg / μL (for UTP-, pUTP-dsRNA) 0.0312-1pg / μL (forN1-Me-pUTP dsRNA), 0.0625-1pg / μL (loogu talagalay 5-OMe-UTP-dsRNA).

Tixgelinta

1. Heerkulka falcelinta TMB iyo waqtiga ayaa muhiim ah, fadlan si adag u xakamee iyaga sida ku cad tilmaamaha.

2. Si loo gaaro dib-u-soo saarid wanaagsan iyo dareenka, dhaqidda saxda ah ee taarikada si meesha looga saaro reagents-ka xad-dhaafka ah ee aan falcelinta lahayn waa lama huraan.

3. Dhammaan reagen-yaasha waa in si fiican loo qaso ka hor inta aan la isticmaalin, lagana fogaado burburka inta lagu jiro muunada ama dib-u-hagaajinta.

4. Haddii kiristaalo ay ka dhex abuurmeen kaydka dhaqdhaqaaga ee xooga leh(20x), diiri ilaa 37℃ oo si tartiib ah isku qas ilaa kiristaalo ay si buuxda u milmeen.

5. Iska ilaali shaybaarada ay ku jiraan Sodium Azide (NaN3), waayo waxay burburin kartaa hawsha HRP taasoo keentay in la qiyaaso qadarka dsRNA.

6. Ka fogow faddaraynta RNase marka la baarayo.

7. Halbeegga/muunada, antibody-ka ogaanshaha iyo SA-HRP ayaa sidoo kale lagu samayn karaa RT iyada oo aan la ruxin, laakiin tani waxay sababi kartaa in dareenka ogaanshaha uu yaraado hal-laab.Xaaladdan oo kale, waxaan kugula talineynaa heerarka UTP iyo pUTP dsRNA waa in laga miiro 2pg/μL, heerarka N1-Me-pUTP dsRNA waa in laga miiro 4pg/μL iyo 5-OMe-UTP dsRNA heerka waa in laga miiro 8pg/μL.Intaa waxaa dheer, ku shub HRP-streptavidin xal shaqeynaya 60min heerkulka qolka.Ha isticmaalin caas gariiriyaha, sababtoo ah gariiriyaha waxaa laga yaabaa inuu keeno natiijo aan sax ahayn.

Natiijada caadiga ah

1. Xogta qalooca caadiga ah

2. Xisaabinta qalooca caadiga ah

3. Qiyaasta ogaanshaha xadhkaha: 0.0312- 1pg/μL