RNA (dsRNA) yamitundu iwiri ya ELISA KIT

Chida ichi ndi Enzyme-Linked Immunosorbent Assay (ELISA) yolumikizana ndi biotin-Streptavidin system, pakuyezera kuchuluka kwa dsRNA yokhala ndi kutalika kopitilira 60 base pairs(bp), mosasamala kanthu za kutsata.Mbaleyi idakutidwa kale ndi anti-dsRNA antibody.dsRNA yomwe ilipo mu zitsanzo imawonjezedwa ndikumangirira ku ma antibodies omwe amakutidwa pazitsime.Kenako biotinylated anti-dsRNA antibody imawonjezedwa ndikumanga ku dsRNA mu zitsanzo.Pambuyo kutsuka, HRP-Streptavidin imawonjezedwa ndikumangirira ku anti-dsRNA antibody ya Biotinylated.Pambuyo pa makulitsidwe, HRP-Streptavidin imatsukidwa.Kenako njira ya TMB gawo lapansi imawonjezedwa ndikupangidwa ndi HRP kuti ipange mtundu wa buluu womwe unasintha kukhala wachikasu pambuyo powonjezera acidic stop solution.Kuchulukana kwachikasu kumayenderana ndi kuchuluka kwa dsRNA komwe kumapezeka mu mbale.Kutsekemera kumayesedwa pa 450 nm.

Kugwiritsa ntchito

Chida ichi ndi choyezera kuchuluka kwa dsRNA yotsalira.

Zida zamagulu

Kusungirako ndi Kukhazikika

1. Pa zida zosagwiritsidwa ntchito: Zida zonse zitha kusungidwa pa 2 ~ 8 ℃ munthawi ya alumali.Kuwala kwamphamvu kuyenera kupewedwa kuti pakhale bata.

2. Kwa zida zomwe zimagwiritsidwa ntchito: Microplate ikatsegulidwa, chonde phimbani zitsime zosagwiritsidwa ntchito ndi chosindikizira mbale ndikubwerera ku thumba la zojambulazo lomwe lili ndi paketi ya desiccant, zip-sindikiza thumba la zojambulazo ndikubwerera ku 2 ~ 8 ℃ mwamsanga mutatha kugwiritsa ntchito.Ma reagents ena amayenera kubwezeredwa ku 2 ~ 8 ℃ posachedwa mukatha kugwiritsa ntchito.

Zinthu Zofunika Koma Zosaperekedwa

1. Microplate reader yokhala ndi fyuluta ya 450±10nm (bwino ngati ingazindikire pa 450 ndi 650 nm wavelength).

2. Microplate shaker.

3. Malangizo opanda RNase ndi machubu a centrifuge.

Operation Flowchart

Musanayambe

1. Bweretsani zigawo zonse za zida ndi zitsanzo kumalo otentha (18-25 ℃) musanagwiritse ntchito.Ngati zida sizidzagwiritsidwa ntchito nthawi imodzi, chonde tengani mizere ndi ma reagents kuti muyesere pano, ndipo siyani mizere yotsalayo ndi ma reagents omwe akufunika.

2. Sambani bafa: chepetsani 40mL ya 20 × centrated kusamba bafa ndi 760mL wa deionized kapena distilled madzi kukonzekera 800mL wa 1× kusamba bafa.

3. Standard: sankhani mwachidule kapena centrifuge yankho la stock musanagwiritse ntchito.Kuchuluka kwa miyezo inayi yoperekedwa ndi 5ng/μL.Pamiyezo ya UTP ndi pUTP dsRNA, chonde chepetsani yankho la masheya ku 1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL ndi STE buffer kuti mujambule pendekera yokhazikika.Pamiyezo ya N1-Me-pUTP dsRNA, chonde chepetsani yankho la masheya ku 2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL ndi STE buffer kuti mujambule mapindikidwe wamba.Pa muyezo wa 5-OMe-UTP dsRNA, chonde chepetsani yankho la masheya kukhala 4,2,1,0.5, 0.25,0.125,0.0625, 0pg/μL ndi STE buffer kuti mujambule mapindikira.Tikupangira kuti milingo itha kuchepetsedwa ngati ma chart awa:

4. Biotinylated detective antibody ndi HRP-streptavidin yogwira ntchito: sankhani mwachidule kapena centrifuge yankho la katundu musanagwiritse ntchito.Achepetseni ku ndende yogwira ntchito ndi dilution buffer.

5. TMB gawo laling'ono: funani mlingo wofunikira wa mankhwalawo ndi nsonga zowawasa ndipo musatayirenso njira yotsalira mu botolo.TMB substate imamva kuwala, osawonetsa gawo la TMB pakuwala kwa nthawi yayitali.

Kugwiritsa ntchito protocol

1. Dziwani kuchuluka kwa mizere yofunikira pakuyesa.Ikani mizere mu mafelemu kuti mugwiritse ntchito.Zotsalira za mbale zotsalira zomwe sizinagwiritsidwe ntchito poyesa izi ziyenera kuikidwanso m'thumba ndi desiccant.Tsekani thumba mwamphamvu kuti musunge mufiriji.

2. Onjezani 100μL iliyonse ya dilution ya muyezo, wopanda kanthu ndi zitsanzo mu zitsime zoyenera.Phimbani ndi chosindikizira mbale.Yambani kwa 1hr kutentha kwa chipinda ndikugwedeza pa 500rpm.Zitsanzozi ziyenera kuchepetsedwa ndi STE buffer kuti zikhale zoyenera kuti ziyesedwe molondola.

3. Sambani sitepe: Aspirate yankho ndi kusamba ndi 250μL kutsuka chotchinga pa chitsime chilichonse ndi kusiya 30s.Tayani chotchinga chotsuka kwathunthu podula mbaleyo papepala loyamwa.Sambani kwathunthu ka 4.

4. Onjezani 100μL ya ma antibody ozindikira a biotinylated m'chitsime chilichonse.Phimbani ndi chosindikizira mbale.Yambani kwa 1hr kutentha kwa chipinda ndikugwedeza pa 500rpm.

5. Bwerezani sitepe yosamba.

6. Onjezani 100μL ya HRP-streptavidin yogwira ntchito mu chitsime chilichonse.Phimbani ndi chosindikizira mbale.Yambani 30min pa firiji ndi kugwedeza pa 500rpm.

7. Bwerezani sitepe yosamba kachiwiri.

8. Onjezani 100μL ya njira ya TMB ya gawo lapansi mu chitsime chilichonse.Phimbani ndi chosindikizira mbale.Yambani kwa mphindi 30 pa RT Tetezani ku kuwala.Madziwo amasanduka buluu powonjezera gawo lapansi.

9. Onjezani 50μL ya njira yoyimitsa mu chitsime chilichonse.Madziwo amasanduka achikasu powonjezera njira yoyimitsa.Kenako thamangani owerenga ma microplate ndikuyesa muyeso pa 450nm nthawi yomweyo.

Kuwerengera Zotsatira

1. Avereji yowerengera kawiri pa mulingo uliwonse, kuwongolera, ndi zitsanzo ndikuchotsa pafupifupi zero kachulukidwe kowoneka bwino.Pangani mipiringidzo yokhazikika ndi kuyamwa pa vertical(Y) axis ndi dsRNA concentration yopingasa(X）axis).

2. Ndibwino kuti muwerenge ndi mapulogalamu opangira ma curve-fitting pakompyuta monga katswiri wa curve 1.3 kapena ELISA Calc mu 5 kapena 4 parameter yosagwirizana ndi mzere.

Kachitidwe

1. Kukhudzika:

malire otsika: ≤ 0.001pg/μL (kwa UTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (kwa 5-OMe-UTP-dsRNA).

malire otsika: 0.0156 pg/μL (ya UTP-, pUTP-dsRNA), 0.0312 pg/μL (ya N1-Me-pUTP-dsRNA), 0.0625 pg/μL (ya 5-OMe-UTP-dsRNA).

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

3. Kuchira: 80% ~ 120%

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

Malingaliro

1. Kutentha kwa TMB ndi nthawi ndizofunikira, chonde ziwongolereni motsatira malangizowo mosamalitsa.

2. Kuti mukwaniritse bwino kuyesa kubwereza ndi kukhudzidwa, kutsuka koyenera kwa mbale kuchotsa ma reagents owonjezera osakhudzidwa ndikofunikira.

3. Onse reagents ayenera kusakaniza bwinobwino isanayambe ntchito ndi kupewa bumbles pa chitsanzo kapena reagents Kuwonjezera.

4. Ngati makhiristo apanga muzosungira zochapira (20x), kutentha mpaka 37 ℃ ndikusakaniza mofatsa mpaka makhiristo asungunuka kwathunthu.

5. Pewani kuyesa kwa zitsanzo zomwe zili ndi Sodium Azide (NaN3), chifukwa zikhoza kuwononga ntchito ya HRP zomwe zimapangitsa kuti tisamawerengere kuchuluka kwa dsRNA.

6. Pewani kuipitsidwa kwa RNase pakuyesa.

7. Muyezo / chitsanzo, ma antibody ozindikira ndi SA-HRP amathanso kuchitidwa ku RT popanda kugwedezeka, koma izi zingayambitse kuzindikira kutsika ndi kamodzi.Pachifukwa ichi, tikupangira kuti miyezo ya UTP ndi pUTP dsRNA ichotsedwe kuchokera ku 2pg/μL, N1-Me-pUTP dsRNA miyezo iyenera kuchepetsedwa kuchokera ku 4pg/μL ndi 5-OMe-UTP dsRNA muyezo uyenera kuchepetsedwa kuchokera ku 8pg/μL.Komanso, incubate HRP-streptavidin ntchito njira 60min firiji.Osagwiritsa ntchito shaker ya flask, chifukwa shaker ya botolo ikhoza kubweretsa zotsatira zolakwika.

Chotsatira chodziwika bwino

1. Deta yokhazikika yopindika

2. Kuwerengera kozungulira kokhazikika

3. Kuzindikira kwa mzere: 0.0312- 1pg / μL