I-RNA ephindwe kabini (dsRNA) ELISA KIT

Le kit yi-Enzyme-Linked Immunosorbent Assay (ELISA) edibanisa ne-biotin-Streptavidin inkqubo, yokulinganisa ubungakanani be-dsRNA kunye nobude obungaphezulu kwe-60 i-pair pairs (bp), kungakhathaliseki ukulandelelana.Ipleyiti ifakwe ngaphambili nge-anti-dsRNA antibody.I-dsRNA ekhoyo kwisampulu yongezwa kwaye ibophelela kwii-antibodies ezigqunywe emaquleni.Kwaye emva koko i-anti-dsRNA i-antibody ye-biotinylated yongezwa kwaye ibophelela kwi-dsRNA kwisampulu.Emva kokuhlamba, i-HRP-Streptavidin yongezwa kwaye ibophelela kwi-antibody ye-Biotinylated anti-dsRNA.Emva kofukamo olungabotshwanga i-HRP-Streptavidin iyahlanjwa.Emva koko isisombululo se-TMB substrate songezwa kwaye sifakwe kwi-HRP ukuvelisa imveliso yombala ozuba oye watshintsha waba tyheli emva kokongeza isisombululo se-acidic stop.Ubuninzi betyheli bulungelelaniswa nesixa ekujoliswe kuso se-dsRNA esifakwe kwipleyiti.I-absorbence ilinganiswa kwi-450 nm.

Isicelo

Le khithi yeyomlinganiselo wobungakanani bentsalela ye-dsRNA.

Amacandelo ekhithi

Ukugcinwa kunye nokuzinza

1. Kwikhithi engasetyenziswanga: Ikhithi yonke inokugcinwa ku-2 ~ 8℃ kubomi beshelufu.Ukukhanya okunamandla kufuneka kugwenywe ukuzinza kokugcina.

2. Kwikiti esetyenzisiweyo: Emva kokuba i-microplate ivuliwe, nceda uvale amaqula angasetyenziswanga kunye ne-plate sealer kwaye ubuyele kwi-foil pouch equkethe ipakethe ye-desiccant, i-zip-seal isikhwama se-foil kwaye ubuyele kwi-2 ~ 8℃ ngokukhawuleza emva kokusetyenziswa.Ezinye ii-reagents kufuneka zibuyiselwe kwi-2 ~ 8℃ ngokukhawuleza emva kokusetyenziswa.

Izinto Eziyimfuneko Kodwa Azibonelelwanga

1. Umfundi weMicroplate one-450±10nm filter (ngcono ukuba unokubhaqa kwi-450 kunye ne-650 nm ubude bobude).

2. Microplate shaker.

3. Iingcebiso ezingenazo i-RNase kunye neetyhubhu ze-centrifuge.

Itshati equkuqelayo yoMsebenzi

Ngaphambi kokuba Uqale

1. Zisa onke amacandelo ekhithi kunye neesampuli kwiqondo lobushushu legumbi (18-25℃) phambi kokusetyenziswa.Ukuba ikhithi ayiyi kusetyenziswa ngexesha elinye, nceda ukhuphe imicu kunye neerejenti zovavanyo lwangoku, kwaye ushiye imicu eseleyo kunye neerejenti zikwimeko efunekayo.

2. Hlamba isithinteli: xuba i-40mL ye-20× egxininisiweyo yesithinteli sokuhlambela nge-760mL yamanzi adiyiniweyo okanye adiliweyo ukulungiselela i-800mL ye-1× yokuhlamba isithinteli.

3. Umgangatho: ngokufutshane spin okanye i-centrifuge isisombululo sesitokhwe ngaphambi kokusetyenziswa.Uxinzelelo lwemigangatho emine enikiweyo yi-5ng/μL.Ngemigangatho ye-UTP kunye ne-pUTP dsRNA, nceda uhlambulule isisombululo sesitokhwe kwi-1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL nge-STE buffer ukuzoba ijika eliqhelekileyo.Ngemigangatho ye-N1-Me-pUTP dsRNA, nceda uhlambulule isisombululo sesitokhwe kwi-2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL nge-STE buffer ukuzoba ijika eliqhelekileyo.Kumgangatho we-5-OMe-UTP dsRNA, nceda uhlambulule isisombululo sesitokhwe kwi-4,2,1,0.5, 0.25,0.125,0.0625, 0pg/μL nge-STE buffer ukuzoba ijika eliqhelekileyo.Sicebisa ukuba imigangatho ingaxutywa njengetshathi ezilandelayo:

4. I-antibody yokufumanisa i-Biotinylated kunye ne-HRP-streptavidin isisombululo sokusebenza: ngokufutshane i-spin okanye i-centrifuge isisombululo sesitokhwe ngaphambi kokusetyenziswa.Zidibanise kugxininiso lokusebenza kunye nesithinteli sokuxutywa.

5. I-TMB substate: nqwenela idosi efunekayo yesisombululo ngeengcebiso ezicociweyo kwaye musa ukulahla isisombululo esishiyekileyo kwi-vial kwakhona.I-TMB substate inovakalelo ekukhanyeni, musa ukuveza i-TMB substrate ekukhanyeni ixesha elide.

Ukusebenzisa iprotocol

1. Qinisekisa inani lemicu efunekayo kuvavanyo.Faka imicu kwiifreyimu ukuze zisetyenziswe.Iipleyiti zeplate eziseleyo ezingasetyenziswanga kolu vavanyo kufuneka ziphinde zipakishwe kwibhegi nge-desiccant.Vala isikhwama ngokuqinileyo ukugcina ifriji.

2. Yongeza i-100μL nganye yedilution yomgangatho, engenanto kunye neesampuli kumaqula afanelekileyo.Gubungela nge-plate sealer.Fudumeza iyure eyi-1 kwiqondo lobushushu begumbi ngokungcangcazela kwi-500rpm.Iisampulu kufuneka zixutywe nge-STE buffer ukuya kugxininiso olufanelekileyo kuvavanyo oluchanekileyo.

3. Inyathelo lokuhlamba: Funa isisombululo kwaye uhlambe nge-250μL ye-buffer yokuhlamba kwiqula ngalinye kwaye uyiyeke ime i-30s.Lahla isithinteli sokuhlamba ngokupheleleyo ngokukrazula ipleyiti kwiphepha elifunxayo.Hlamba ngokupheleleyo amaxesha ama-4.

4. Yongeza i-100μL yesisombululo esisebenzayo sokuxilongwa kwe-antibody kwiqula ngalinye.Gubungela nge-plate sealer.Fudumeza iyure eyi-1 kwiqondo lobushushu begumbi ngokungcangcazela kwi-500rpm.

5. Phinda uhlambe inyathelo.

6. Yongeza i-100μL yesisombululo esisebenzayo se-HRP-streptavidin kwiqula ngalinye.Gubungela nge-plate sealer.Fudumeza i-30min kwiqondo lobushushu begumbi ngokungcangcazela kwi-500rpm.

7. Phinda uhlambe kwakhona.

8. Yongeza i-100μL yesisombululo se-TMB substrate kwiqula ngalinye.Gubungela nge-plate sealer.Fuka i-30 min kwi-RT Khusela ekukhanyeni.Ulwelo luya kujika lube luhlaza okwesibhakabhaka ngokongezwa kwesisombululo se-substrate.

9. Yongeza i-50μL yesisombululo sokumisa kwiqula ngalinye.Ulwelo luya kuba tyheli ngokongezwa kwesisombululo sokumisa.Emva koko sebenzisa i-microplate reader kwaye uqhube umlinganiselo kwi-450nm ngoko nangoko.

Ukubalwa kweZiphumo

1. I-avareji yofundo oluphindwe kabini kumgangatho ngamnye, ulawulo, kunye neesampulu kwaye uthabathe umndilili we-zero standard optical density.Yakha igophe elisemgangathweni ngokufunxa kwi-axis ethe nkqo(Y) kunye nogxininiso lwe-dsRNA kwindawo ethe tye(X）umgca we-axis).

2. Kucetyiswa ukubala kubalo nge-software esekelwe kwi-curve-fitting software efana ne-curve expert 1.3 okanye i-ELISA Calc kwi-5 okanye 4 parameter non-linear fit model.

Ukusebenza

1. Uvakalelo:

umda ophantsi wokufumanisa: ≤ 0.001pg/μL (ye-UTP-, i-pUTP-, i-N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (ye-5-OMe-UTP-dsRNA).

umda osezantsi wobungakanani: 0.0156 pg/μL (ye-UTP-, i-pUTP-dsRNA), 0.0312 pg/μL (ye-N1-Me-pUTP-dsRNA), 0.0625 pg/μL (ye-5-OMe-UTP-dsRNA).

2. Ukuchaneka: I-CV ye-Intra-Assay ≤10%, i-CV ye-Inter-Assay ≤10%

3. Ukubuyisela kwakhona: 80% ~ 120%

4.Umgca:0.0156-0.5pg/μL(forUTP-,pUTP-dsRNA)0.0312-1pg/μL(forN1-Me-pUTP dsRNA),0.0625-1pg/μL(ye-5-OMe-UTP-dsRNA).

Iingqwalasela

1. TMB reaction lobushushu kunye nexesha kubalulekile, nceda uzilawule ngokomyalelo ngokungqongqo.

2. Ukuze kuphunyezwe ukuveliswa kwakhona kwe-assay kunye novelwano, ukuhlamba ngokufanelekileyo kweeplate ukususa i-reagents engaphezulu engaphendulwanga ibalulekile.

3. Zonke ii-reagents kufuneka zixutywe ngokucokisekileyo ngaphambi kokusetyenziswa kwaye zigweme ama-bumbles ngexesha lesampuli okanye i-reagents yokudibanisa.

4. Ukuba iikristale zenziwe kwisithinteli sokuhlamba esigxininisiweyo (20x), shushu ukuya kwi-37℃ kwaye uxube ngobunono de iikristale zinyibilike ngokupheleleyo.

5. Gwema ukuvavanya iisampuli eziqulethe i-Sodium Azide (NaN3), njengoko inokutshabalalisa umsebenzi we-HRP obangela ukuqikelelwa ngaphantsi kwesixa se-dsRNA.

6. Kuphephe ukosuleleka kwe-RNase ngexesha lovavanyo.

7. Umgangatho / isampuli, i-antibody yokufumanisa kunye ne-SA-HRP nayo inokuqhutywa kwi-RT ngaphandle kokungcangcazela, kodwa oku kunokubangela ukuba uvakalelo lokufumanisa luhla ngokuphindwe kabini.Kule meko, sincoma imigangatho ye-UTP kunye ne-pUTP dsRNA kufuneka ihlanjululwe ukusuka kwi-2pg/μL, imigangatho ye-N1-Me-pUTP dsRNA kufuneka ihlanjululwe ukusuka kwi-4pg/μL kunye ne-5-OMe-UTP dsRNA umgangatho kufuneka uhlanjululwe ukusuka kwi-8pg/μL.Ukongeza, faka isisombululo esisebenzayo se-HRP-streptavidin se-60min kwiqondo lokushisa.Ungasebenzisi i-flask shaker, kuba i-flask shaker inokubangela iziphumo ezingachanekanga.

Isiphumo esiqhelekileyo

1. Idatha yegophe eqhelekileyo

2. Ukubalwa kwegophe eqhelekileyo

3. Uluhlu lokukhangela umgca: 0.0312- 1pg / μL