Yakapetwa kaviri RNA (dsRNA) ELISA KIT

Iyi kit iEnzyme-Linked Immunosorbent Assay (ELISA) yakabatana nebiotin-Streptavidin system, yekuyera kuyerwa kwedsRNA nehurefu huri pamusoro pemakumi matanhatu base pairs(bp), zvisinei nekutevedzana.Iyo ndiro yakambovharwa ne anti-dsRNA antibody.dsRNA iripo mumuenzaniso inowedzerwa uye inosungirirwa kune masoja ekudzivirira chirwere akaputirwa pamatsime.Uyezve biotinylated anti-dsRNA antibody inowedzerwa uye inosungirira ku dsRNA mumuenzaniso.Mushure mekugeza, HRP-Streptavidin inowedzerwa uye inosunga kune Biotinylated anti-dsRNA antibody.Mushure mekuita incubation isina kusungwa HRP-Streptavidin inogezwa.Ipapo TMB substrate mhinduro inowedzerwa uye inonatswa neHRP kugadzira yebhuruu ruvara chigadzirwa chakashanduka kuita yero mushure mekuwedzera acidic stop solution.Density yeyero inoenderana nehuwandu hwechinangwa che dsRNA chakabatwa mundiro.Iyo absorbance inoyerwa pa450 nm.

Application

Iyi kit ndeyekuyerwa kuyerwa kwezvakasara dsRNA.

Kit zvikamu

Kuchengetedza uye Kugadzikana

1. Yekiti isina kushandiswa: Kiti yese inogona kuchengetwa pa2 ~ 8 ℃ muhupenyu hwesherufu.Chiedza chakasimba chinofanira kudziviswa kuitira kugadzikana kwekuchengetedza.

2. Yekiti yakashandiswa: Kana iyo microplate yavhurwa, tapota vhara matsime asina kushandiswa neplate sealer uye dzokera kune foil pouch ine desiccant pack, zip-seal foil pouch uye dzokera ku2 ~ 8 ℃ nokukurumidza sezvinobvira mushure mekushandisa.Mamwe mareagents anofanirwa kudzoserwa ku2 ~ 8 ℃ nekukurumidza sezvinobvira mushure mekushandisa.

Zvokushandisa Zvinodiwa Asi Zvisina Kupiwa

1. Microplate reader ine 450±10nm sefa (zviri nani kana ichigona kuona pa450 uye 650 nm wavelength).

2. Microplate shaker.

3. RNase-isina mazano uye centrifuge tubes.

Operation Flowchart

Usati Watanga

1. Hunza zvese zvikamu zvekiti uye samples kune tembiricha yekamuri (18-25 ℃) usati washandisa.Kana iyo kiti ikasazoshandiswa kumusoro panguva imwe chete, ndapota ingotora mitsetse uye mareagents ekuedza kwazvino, uye siya akasara mitsetse nemareagents ari muchimiro chinodiwa.

2. Wash buffer: diluter 40mL ye20× concentrated wash buffer ne 760mL yemvura yakasvibiswa kana yakasvibiswa kugadzirira 800mL ye1× wash buffer.

3. Chiyero: muchidimbu spin kana centrifuge mushonga wemasheya usati washandisa.Iko kusungirirwa kwezviyero zvina zvakapihwa ndeye 5ng/μL.Nezve UTP uye pUTP dsRNA zviyero, ndapota dzikisa mhinduro yestock ku1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL ine STE buffer kudhirowa yakajairwa curve.Nezve N1-Me-pUTP dsRNA zviyero, ndapota dzikisa mhinduro yemasheya ku2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL ine STE buffer kudhirowa yakajairwa curve.Kune 5-OMe-UTP dsRNA yakajairwa, ndapota dzikisa mhinduro yemasheya ku4,2,1,0.5, 0.25,0.125,0.0625, 0pg/μL ine STE buffer kudhirowa yakajairwa curve.Isu tinokurudzira zviyero zvinogona kuderedzwa seanotevera machati:

4. Biotinylated inoona antibody uye HRP-streptavidin kushanda mhinduro: pfupi pfupi spin kana centrifuge mhinduro yemasheya isati yashandiswa.Dilute ivo kune iyo yekushanda yekusangana ine dilution buffer.

5. TMB substate: shuvira mwero unodiwa wemushonga nemazano ane sterilized uye usakanda mushonga wasara mubhodhoro zvakare.TMB substate inonzwa kuchiedza, usaratidze TMB substrate kuchiedza kwenguva yakareba.

Kushandisa protocol

1. Sarudza nhamba yezvidimbu zvinodiwa pakuongorora.Isa mitsetse mumafuremu kuti ishandiswe.Yakasara yeplate mitsetse isina kushandiswa muyedzo iyi inofanirwa kudzokororwa mubhegi ne desiccant.Vhara bhegi zvakasimba kuitira kuchengetedza firiji.

2. Wedzera 100μL imwe neimwe ye dilutions yezviyero, isina chinhu uye sampuli mumatsime akakodzera.Fukidza neplate sealer.Chengetedza 1hr pakamuri tembiricha uchizunguza pa500rpm.Iwo masampuli anofanirwa kuderedzwa neSTE buffer kune yakakodzera kusungwa kweiyo assay.

3. Geza nhanho: Aspirate solution wogeza ne 250μL wash buffer patsime rega rega wosiya ikamira kwe30s.Rasa wash buffer zvachose nekutsemura ndiro pabepa rinopinza.Wacha zvachose ka4.

4. Wedzera 100μL yebiotinylated inoona masoja ekudzivirira chirwere anoshanda mutsime rega rega.Fukidza neplate sealer.Chengetedza 1hr pakamuri tembiricha uchizunguza pa500rpm.

5. Dzokorora danho rekugeza.

6. Wedzera 100μL yeHRP-streptavidin inoshanda mhinduro mutsime rimwe nerimwe.Fukidza neplate sealer.Chengetedza 30min pakamuri tembiricha nekuzunguza pa500rpm.

7. Dzokorora danho rekugeza zvakare.

8. Wedzera 100μL yeTMB substrate solution mutsime rega rega.Fukidza neplate sealer.Isa maminitsi makumi matatu paRT Dzivirira kubva kuchiedza.Iyo mvura inoshanduka bhuruu nekuwedzera kwe substrate solution.

9. Wedzera 50μL yemhinduro yekumisa mutsime rega rega.Iyo mvura inoshanduka yero nekuwedzera kwekumisa mhinduro.Wobva wamhanya iyo microplate muverengi uye itisa kuyerwa pa450nm nekukasira.

Calculation of Results

1. Avhareji yekuverenga kwakapetwa pachiyero chega chega, kutonga, uye masampuli uye kubvisa avhareji zero standard optical density.Gadzira curve yakajairwa ine kunyura pane yakatwasuka(Y) axis uye dsRNA concentration pane yakachinjika(X）axis.

2. Zvinokurudzirwa kuita kuverenga nekombuta-based curve-fitting software senge curve nyanzvi 1.3 kana ELISA Calc mune 5 kana 4 parameter non-linear fit model.

Performance

1. Sensitivity:

muganho wakaderera wekuona: ≤ 0.001pg/μL (yeUTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (ye5-OMe-UTP-dsRNA).

muganho wakaderera wehuwandu:0.0156 pg/μL (yeUTP-, pUTP-dsRNA), 0.0312 pg/μL (yeN1-Me-pUTP-dsRNA), 0.0625 pg/μL (ye5-OMe-UTP-dsRNA).

2. Kururamisa: CV yeIntra-Assay ≤10%, CV yeInter-Assay ≤10%

3. Kuwanazve:80%~120%

4.Linearity:0.0156-0.5pg/μL(forUTP-,pUTP-dsRNA)0.0312-1pg/μL(forN1-Me-pUTP dsRNA),0.0625-1pg/μL(ye5-OMe-UTP-dsRNA).

Pfungwa

1. TMB reaction tembiricha uye nguva yakakosha, ndapota vadzore zvinoenderana nekuraira kwakasimba.

2. Kuti uwane kubereka kwakanaka kwekuongorora uye kunzwisiswa, kushambidza kwakakodzera kweplateti kubvisa zvakawandisa zvisingagadziriswi reagents zvakakosha.

3. Zvose zvinogadzirisa zvinofanira kusanganiswa zvakakwana zvisati zvashandiswa uye kudzivisa mabhubhu panguva yemuenzaniso kana reagents kuwedzera.

4. Kana makristasi akaumbwa mubhafa yekuwachisa yakadzika(20x), inodziya kusvika 37℃ uye sanganisa zvinyoro kusvika makristasi anyungudika zvachose.

5. Dzivisa kuongororwa kwemasampuli ane Sodium Azide (NaN3), sezvo inogona kuparadza basa reHRP rinokonzera pasi-kufungidzirwa kwehuwandu hwe dsRNA.

6. Dzivisa kusvibiswa kweRNase panguva yekuedzwa.

7. Muyero / sampuli, antibody yekuona uye SA-HRP inogonawo kuitwa paRT pasina kuzununguka, asi izvi zvinogona kukonzera kunzwisiswa kwekunzwa kuderera nekamwe-kamwe.Panyaya iyi, isu tinokurudzira UTP uye pUTP dsRNA zviyero zvinofanirwa kuderedzwa kubva pa2pg/μL, N1-Me-pUTP dsRNA zviyero zvinofanirwa kuderedzwa kubva pa4pg/μL uye 5-OMe-UTP dsRNA standard inofanira kuderedzwa kubva pa8pg/μL.Mukuwedzera, incubate HRP-streptavidin kushanda mhinduro ye60min pakamuri tembiricha.Usashandise flask shaker, nekuti flask shaker inogona kukonzeresa mhedzisiro.

Mubairo chaiwo

1. Standard curve data

2. Standard curve calculation

3. Liner yekuona huwandu: 0.0312- 1pg / μL