RNA ikubye kabiri (dsRNA) ELISA KIT

Iki gikoresho ni Enzyme-Ihuza Immunosorbent Assay (ELISA) ifatanije na sisitemu ya biotin-Streptavidin, kugirango bapime ingano ya dsRNA ifite uburebure buri hejuru ya 60 shingiro (bp), hatitawe ku ruhererekane.Isahani yashizwemo mbere na antibody anti-dsRNA.dsRNA igaragara murugero yongeweho kandi ihuza antibodies zometse kumariba.Hanyuma antibody ya biotinylated anti-dsRNA yongeweho kandi ihuza dsRNA murugero.Nyuma yo gukaraba, HRP-Streptavidin yongewemo kandi ihuza na antibody ya Biotinylated anti-dsRNA.Nyuma ya incubation idahuza HRP-Streptavidin yogejwe.Noneho igisubizo cya TMB substrate cyongeweho kandi kigatangizwa na HRP kugirango gitange ibicuruzwa byamabara yubururu byahindutse umuhondo nyuma yo kongeramo aside ihagarika.Ubucucike bwumuhondo buringaniye nintego ya dsRNA yafashwe mu isahani.Kwinjira byapimwe kuri 450 nm.

Gusaba

Iki gikoresho nigipimo cyo gupima ingano ya dsRNA isigaye.

Ibigize ibikoresho

Kubika no Guhagarara

1. Kubikoresho bidakoreshwa: Igikoresho cyose gishobora kubikwa kuri 2 ~ 8 ℃ mubuzima bwubuzima.Umucyo ukomeye ugomba kwirindwa kugirango ubike neza.

2. Kubikoresho byakoreshejwe: Microplate imaze gukingurwa, nyamuneka utwikire amariba adakoreshwa hamwe na kashe ya plaque hanyuma usubire mu mufuka wa file urimo ipaki ya desiccant, zip-kashe umufuka wa file hanyuma usubire kuri 2 ~ 8 ℃ vuba bishoboka nyuma yo kuyikoresha.Izindi reagent zigomba gusubizwa kuri 2 ~ 8 ℃ byihuse nyuma yo gukoreshwa.

Ibikoresho bisabwa ariko ntibitangwa

1. Umusomyi wa Microplate hamwe na 450 ± 10nm muyunguruzi (nibyiza niba ushobora kumenya kuri 450 na 650 nm yumurambararo).

2. Shitingi ya Microplate.

3. Inama zidafite RNase hamwe na trub ya centrifuge.

Igikorwa cya Flowchart

Mbere yuko Utangira

1. Zana ibikoresho byose hamwe nicyitegererezo mubushyuhe bwicyumba (18-25 ℃) mbere yo gukoresha.Niba ibikoresho bitazakoreshwa mugihe kimwe, nyamuneka fata gusa imirongo na reagent kugirango ugerageze ubungubu, hanyuma usige imirongo isigaye na reagent muburyo bukenewe.

2. Gukaraba buffer: koresha 40mL ya 20 × ya bffer yo gukaraba hamwe na 760mL y'amazi ya deionisation cyangwa yatoboye kugirango utegure 800mL ya 1 × yoza.

3. Ibisanzwe: kuzunguruka muri make cyangwa centrifuge igisubizo cyibigega mbere yo gukoresha.Ubwinshi bwibipimo bine byatanzwe ni 5ng / μL.Kubipimo bya UTP na pUTP dsRNA, nyamuneka koresha igisubizo cyimigabane kuri 1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg / μL hamwe na buffer ya STE gushushanya umurongo usanzwe.Kubipimo bya N1-Me-pUTP dsRNA, nyamuneka koresha igisubizo cyibicuruzwa kuri 2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg / μL hamwe na buffer ya STE gushushanya umurongo usanzwe.Kuri 5-OMe-UTP dsRNA isanzwe, nyamuneka koresha igisubizo cyimigabane kuri 4.2,1,0.5, 0.25,0.125,0.0625, 0pg / μL hamwe na buffer ya STE gushushanya umurongo usanzwe.Turasaba ko ibipimo bishobora kugabanywa nkimbonerahamwe ikurikira:

4. Biotinylated detection antibody na HRP-streptavidin igisubizo cyakazi: kuzenguruka gato cyangwa centrifuge igisubizo cyibigega mbere yo kubikoresha.Kubishira kumurongo wibikorwa hamwe na dilution buffer.

5. TMB isimburana: wifuze dosiye ikenewe yumuti hamwe ninama zifatika kandi ntuzongere guta igisubizo gisigaye muri vial.Substate ya TMB yunvikana kumucyo, ntugaragaze substrate ya TMB kumucyo igihe kirekire.

Ukoresheje protocole

1. Menya umubare wibice bisabwa kugirango usuzumwe.Shyiramo imirongo kumurongo kugirango ukoreshe.Ibisigazwa by'ibisahani bisigaye bidakoreshwa muri ubu bushakashatsi bigomba gusubirwamo mu mufuka hamwe na desiccant.Funga igikapu neza kugirango ubike firigo.

2. Ongeramo 100μL buri kimwe cyo guhinduranya bisanzwe, ubusa hamwe nicyitegererezo mumariba akwiye.Gupfukirana icyapa.Shyiramo 1hr mubushyuhe bwicyumba hamwe no kunyeganyega 500rpm.Ibyitegererezo bigomba kuvangwa na STE buffer kugirango bibe byibanze kugirango bisuzumwe neza.

3. Karaba intambwe: Saba igisubizo hanyuma ukarabe hamwe na 250μL yoza buffer kuri buri riba hanyuma ureke bihagarare 30.Kureka gukaraba neza ukoresheje isahani ku mpapuro zinjira.Gukaraba rwose inshuro 4.

4. Ongeramo 100μL ya biotinylated detection antibody ikora igisubizo muri buri riba.Gupfukirana icyapa.Shyiramo 1hr mubushyuhe bwicyumba hamwe no kunyeganyega 500rpm.

5. Subiramo intambwe yo gukaraba.

6. Ongeramo 100μL ya HRP-streptavidin igisubizo gikora muri buri riba.Gupfukirana icyapa.Shyiramo 30min mubushyuhe bwicyumba hamwe no kunyeganyega 500rpm.

7. Ongera usubiremo intambwe yo gukaraba.

8. Ongeramo 100μL ya TMB substrate igisubizo muri buri riba.Gupfukirana icyapa.Incubate kuminota 30 kuri RT Kurinda urumuri.Amazi azahinduka ubururu hiyongereyeho igisubizo cya substrate.

9. Ongeramo 50μL yo guhagarika igisubizo muri buri riba.Amazi azahinduka umuhondo hiyongereyeho igisubizo cyo guhagarika.Noneho koresha microplate umusomyi hanyuma ukore ibipimo kuri 450nm ako kanya.

Kubara Ibisubizo

1. Gereranya ibyasomwe byombi kuri buri gipimo, kugenzura, hamwe nicyitegererezo hanyuma ukuremo impuzandengo ya zeru ya optique yuzuye.Kubaka umurongo usanzwe hamwe no kwinjiza kumurongo uhagaze (Y) hamwe na dsRNA yibanze kuri horizontal (X） axis.

2. Birasabwa gukora ibarwa hamwe na mudasobwa ishingiye kuri mudasobwa ikwiranye na software nkumuhanga wu murongo 1.3 cyangwa ELISA Calc muburyo bwa 5 cyangwa 4 muburyo butari umurongo.

Imikorere

1. Ibyiyumvo:

ntarengwa yo gutahura: ≤ 0.001pg / μL (kuri UTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg / μL (kuri 5-OMe-UTP-dsRNA).

ntarengwa yo kubara: 0.0156 pg / μL (kuri UTP-, pUTP-dsRNA), 0.0312 pg / μL (kuri N1-Me-pUTP-dsRNA), 0.0625 pg / μL (kuri 5-OMe-UTP-dsRNA).

2. Icyitonderwa: CV ya Intra-Assay ≤10%, CV ya Inter-Assay ≤10%

3. Gukira: 80% ~ 120%

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

Ibitekerezo

1. Ubushyuhe bwa TMB nibihe birakomeye, nyamuneka ubigenzure ukurikije amabwiriza neza.

2. Kugirango ugere ku myororokere myiza no kwiyumvisha ibintu, gukaraba neza amasahani kugirango ukureho reagent zirenze urugero ni ngombwa.

3. Reagent zose zigomba kuvangwa neza mbere yo gukoresha no kwirinda gutitira mugihe cyicyitegererezo cyangwa reagent wongeyeho.

4. Niba kristu zarakozwe mumashanyarazi yogejwe (20x), shyushya kuri 37 ℃ hanyuma uvange witonze kugeza kristu zashonze burundu.

5. Irinde gusuzuma ingero zirimo Sodium Azide (NaN3), kuko ishobora gusenya ibikorwa bya HRP bigatuma hatagereranywa umubare wa dsRNA.

6. Irinde kwanduza RNase mugihe cyo gusuzuma.

7. Igipimo / icyitegererezo, antibody yo gutahura na SA-HRP nacyo gishobora gukorerwa kuri RT nta guhungabana, ariko ibyo birashobora gutuma sensibilité yo kugabanuka igabanuka inshuro imwe.Kuri uru rubanza, turasaba ko ibipimo bya UTP na pUTP dsRNA bigomba kuvomwa kuva 2pg / μL, N1-Me-pUTP dsRNA bigomba kuvangwa kuva 4pg / μL naho 5-OMe-UTP dsRNA bigomba kuvangwa kuva 8pg / μL.Wongeyeho, incubate HRP-streptavidin igisubizo cyakazi kuri 60min mubushyuhe bwicyumba.Ntukoreshe flask shaker, kuko flask shaker irashobora kuvamo ibisubizo bidahwitse.

Ibisubizo bisanzwe

1. Ibyatanzwe bisanzwe

2. Kubara bisanzwe

3. Urutonde rwo gutahura: 0.0312- 1pg / μL