RNA kaulua (dsRNA) ELISA KIT

ʻO kēia kit he Enzyme-Linked Immunosorbent Assay (ELISA) e hui pū ana me ka biotin-Streptavidin system, no ke ana nui o ka dsRNA me ka lōʻihi ma luna o 60 base pairs(bp), me ka ʻole o ke kaʻina.Ua uhi mua ʻia ka pā me ka anti-dsRNA antibody.Hoʻohui ʻia ka dsRNA i loko o ka laʻana a hoʻopaʻa ʻia i nā antibodies i uhi ʻia ma nā pūnāwai.A laila hoʻohui ʻia ka anti-dsRNA antibody biotinylated a hoʻopaʻa ʻia i ka dsRNA i ka hāpana.Ma hope o ka holoi ʻana, hoʻohui ʻia ʻo HRP-Streptavidin a hoʻopaʻa ʻia i ka antibody anti-dsRNA Biotinylated.Ma hope o ka incubation unbound HRP-Streptavidin holoi ʻia.A laila hoʻohui ʻia ka hopena substrate TMB a hoʻopaʻa ʻia e HRP e hana i kahi huahana kala polū i hoʻololi ʻia i melemele ma hope o ka hoʻohui ʻana i ka hopena hoʻomaha acidic.Hoʻohālikelike ka nui o ka melemele i ka nui o ka dsRNA i hopu ʻia ma ka pā.ʻIke ʻia ka absorbance ma 450 nm.

Palapala noi

ʻO kēia pahu no ke ana nui o ke koena dsRNA.

Nā ʻāpana pahu

Ka Waihona a me ka Paʻa

1. No ka pahu i hoʻohana ʻole ʻia: Hiki ke mālama ʻia ka pahu holoʻokoʻa ma 2 ~ 8 ℃ i ke ola ʻana.Pono e pale ʻia ka mālamalama ikaika no ka mālama ʻana.

2. No ka pahu i hoʻohana ʻia: Ke wehe ʻia ka microplate, e ʻoluʻolu e uhi i nā pūnāwai i hoʻohana ʻole ʻia me ka sealer plate a hoʻihoʻi i ka ʻeke pepa i loaʻa ka pahu desiccant, hoʻopaʻa zip-i ka ʻeke pepa a hoʻihoʻi i ka 2 ~ 8 ℃ i ka hikiwawe ma hope o ka hoʻohana ʻana.Pono e hoʻihoʻi ʻia nā reagents ʻē aʻe i 2 ~ 8 ℃ i ka wā hiki loa ma hope o ka hoʻohana ʻana.

Pono nā mea hana akā ʻaʻole i hoʻolako ʻia

1. Heluhelu microplate me ka kānana 450±10nm (ʻoi aku ka maikaʻi inā hiki ke ʻike ma 450 a me 650 nm ka lōʻihi).

2. Mea lulu microplate.

3. Nā ʻōlelo aʻoaʻo RNase ʻole a me nā paipu centrifuge.

Ka Papa Hana

Ma mua o kou hoʻomaka ʻana

1. E lawe mai i na mea a pau a me na la'ana i ka lumi wela (18-25 ℃) ma mua o ka ho'ohana 'ana.Inā ʻaʻole hoʻohana ʻia ka pahu i ka manawa hoʻokahi, e ʻoluʻolu e lawe wale i waho i nā ʻāpana a me nā reagents no ka hoʻokolohua o kēia manawa, a waiho i nā ʻāpana a me nā reagents i koe i ke kūlana pono.

2. Holoi pale: dilute 40mL o 20x concentrated holoi holoi me 760mL o deionized a distilled wai e hoomakaukau i 800mL o 1x holoi holoi.

3. Maʻamau: wili pōkole a centrifuge i ka hopena waiwai ma mua o ka hoʻohana ʻana.ʻO ka manaʻo o nā kūlana ʻehā i hāʻawi ʻia ʻo 5ng / μL.No nā kūlana UTP a me pUTP dsRNA, e ʻoluʻolu e hoʻoheheʻe i ka solution stock i 1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL me STE buffer e kaha i ka pihi maʻamau.No nā maʻamau N1-Me-pUTP dsRNA, e ʻoluʻolu e hoʻoheheʻe i ka solution stock i ka 2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL me ka STE buffer e kaha i ka pihi maʻamau.No ka maʻamau 5-OMe-UTP dsRNA, e ʻoluʻolu e hoʻoheheʻe i ka solution stock i 4,2,1,0.5, 0.25,0.125,0.0625, 0pg/μL me STE buffer e kaha i ka pihi maʻamau.Manaʻo mākou e hiki ke hoʻoheheʻe ʻia nā maʻamau e like me nā pakuhi:

4. Biotinylated detection antibody a me HRP-streptavidin solution working solution: wili pōkole a centrifuge i ka solution stock before use.E hoʻoheheʻe iā lākou i ka ʻike hana me ka dilution buffer.

5. TMB substate: aspirate i ka mea e pono ai o ka hoʻonā me nā ʻōlelo aʻoaʻo sterilized a mai hoʻolei hou i ke koena i loko o ka hue.Hoʻopili ka TMB substate i ka mālamalama, mai hōʻike i ka substrate TMB i ke kukui no ka manawa lōʻihi.

Ke hoʻohana nei i ka protocol

1. E hoʻoholo i ka helu o nā ʻāpana e pono ai no ka hoʻāʻo.E hoʻokomo i nā ʻāpana i loko o nā pā no ka hoʻohana ʻana.Pono e hoʻopaʻa hou ʻia nā ʻāpana pā i hoʻohana ʻole ʻia i loko o ka ʻeke me ka desiccant.E pani paʻa i ka ʻeke no ka waiho ʻana i loko o ka pahu hau.

2. E hoʻohui i ka 100μL i kēlā me kēia dilutions o ka maʻamau, blank a me nā laʻana i loko o nā pūnāwai kūpono.E uhi me ka mea hoʻopaʻa pā.Incubate no 1hr ma ka lumi wela me ka lulu ma 500rpm.Pono e hoʻoheheʻe ʻia nā laʻana me ka STE buffer i ka ʻike kūpono no ka ʻike pololei.

3. Holoi ʻanuʻu: Aspirate i ka hopena a holoi me 250μL holoi holoi i kēlā me kēia pūnāwai a waiho ia no 30s.E hoʻolei loa i ka mea holoi holoi ma ka hoʻopaʻa ʻana i ka pā ma luna o ka pepa ʻoluʻolu.Holoi loa 4 manawa.

4. Hoʻohui i 100μL o ka biotinylated detection antibody working solution i loko o kēlā me kēia pūnāwai.E uhi me ka mea hoʻopaʻa pā.Incubate no 1hr ma ka lumi wela me ka lulu ma 500rpm.

5. E hana hou i ka holoi holoi.

6. E hoʻohui i 100μL o ka HRP-streptavidin hana hana i loko o kēlā me kēia luawai.E uhi me ka mea hoʻopaʻa pā.Incubate no 30min ma ka lumi wela me ka lulu ma 500rpm.

7. E hana hou i ka holoi holoi.

8. E hoʻohui i 100μL o ka TMB substrate solution i loko o kēlā me kēia pūnāwai.E uhi me ka mea hoʻopaʻa pā.Hoʻomoʻa no 30 min ma RT Pale mai ka mālamalama.E lilo ka wai i polū ma ka hoʻohui ʻana i ka hopena substrate.

9. E hoʻohui i 50μL o ka hopena hoʻomaha i kēlā me kēia pūnāwai.E melemele ka wai ma ka hoʻohui ʻana i ka solution stop.A laila holo i ka mea heluhelu microplate a hana i ke ana ma 450nm koke.

Heluhelu i nā hopena

1. E awelika i nā heluhelu pālua no kēlā me kēia maʻamau, ka mana, a me nā laʻana a e unuhi i ka awelika zero standard optical density.E kūkulu i ka pihi maʻamau me ka hoʻopaʻa ʻana ma ke koʻi kū pololei (Y) a me ka neʻe ʻana o ka dsRNA ma ka axis (X) axis.

2. Manaʻo ʻia e hana i ka helu ʻana me ka lako kamepiula e pili ana i ka curve-fitting software e like me curve expert 1.3 a i ʻole ELISA Calc i loko o kahi kumu hoʻohālike kūpono ʻole linear 5 a 4 paha.

hana

1. Naʻauao:

palena haʻahaʻa o ka ʻike: ≤ 0.001pg/μL (no UTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (no 5-OMe-UTP-dsRNA).

palena haʻahaʻa o ka quantitation:0.0156 pg/μL (no UTP-, pUTP-dsRNA),0.0312 pg/μL (no N1-Me-pUTP-dsRNA),0.0625 pg/μL (no 5-OMe-UTP-dsRNA).

2. Ka pololei: CV o Intra-Assay ≤10%, CV o Inter-Assay ≤10%

3. Hoʻihoʻi hou: 80% ~ 120%

4.Linearity:0.0156-0.5pg/μL(noUTP-,pUTP-dsRNA)0.0312-1pg/μL(noN1-Me-pUTP dsRNA),0.0625-1pg/μL(no 5-OMe-UTP-dsRNA).

Noonoo

1. He koʻikoʻi ka wela o ka hopena TMB a me ka manawa, e ʻoluʻolu e hoʻomalu iā lākou e like me ke aʻo ʻana.

2. I mea e loaʻa ai ka maikaʻi o ka assay reproducibility a me ka naʻau, ʻo ka holoi pono ʻana i nā papa e wehe i nā reagents i hoʻoneʻe ʻole ʻia he mea nui.

3. Pono e hui maika'i nā reagents a pau ma mua o ka ho'ohana 'ana a pale aku i nā pu'upu'u i ka wā o ka la'ana a i 'ole ka ho'ohui 'ana.

4. Inā ua hoʻokumu ʻia nā kristal i loko o ka pahu holoi holoi (20x), mehana i 37 ℃ a hui mālie a hiki i ka hemo ʻana o nā kristal.

5. E pale i ka ho'āʻo ʻana i nā laʻana i loaʻa ka Sodium Azide (NaN3), no ka mea hiki iā ia ke hoʻopau i ka hana HRP e hopena i ka manaʻo haʻahaʻa o ka nui o dsRNA.

6. E pale i ka haumia RNase i ka wā e ho'āʻo ai.

7. Hiki ke alaka'i 'ia ka ma'amau/la'ana, antibody detection a me SA-HRP ma RT me ka lulu 'ole, akā hiki ke ho'emi 'ia ka 'ike 'ike ma ka pa'kahi.No kēia hihia, makemake mākou e hoʻoheheʻe ʻia nā kūlana UTP a me pUTP dsRNA mai 2pg/μL, pono e hoʻoheheʻe ʻia nā maʻamau N1-Me-pUTP dsRNA mai 4pg/μL a me 5-OMe-UTP dsRNA maʻamau e hoʻoheheʻe ʻia mai 8pg/μL.Eia kekahi, e hoʻokomo i ka hopena hana HRP-streptavidin no 60min ma ka lumi wela.Mai hoʻohana i ka hoʻoluliluli pahu, no ka mea, hiki ke hopena i ka hopena pololei.

Ka hopena maʻamau

1. ʻIkepili piko maʻamau

2. Helu helu ma'amau

3. Laina ike laina:0.0312- 1pg/μL