RNA (dsRNA) ELISA KIT nwere eriri okpukpu abụọ

Ngwa a bụ Enzyme-Linked Immunosorbent Assay (ELISA) jikọtara ya na sistemu biotin-Streptavidin, maka nha nha nke dsRNA nwere ogologo karịa 60 base pairs(bp), n'agbanyeghị usoro ya.Ejikwa mgbochi dsRNA kpuchie efere ahụ.A na-agbakwunye dsRNA dị na nlele ma na-ejikọta ya na ọgwụ mgbochi ndị ekpuchiri n'olulu mmiri.Mgbe ahụ, a na-agbakwunye ọgwụ mgbochi dsRNA biotinylated wee jikọta na dsRNA na nlele.Mgbe ịsachara, a na-agbakwunye HRP-Streptavidin ma jikọta na mgbochi dsRNA Biotinylated.Mgbe nnabata ahụ gwụchara, a na-asachapụ HRP-Streptavidin.Mgbe ahụ, a na-agbakwunye ihe ngwọta TMB na catalyzed site na HRP iji mepụta ngwaahịa na-acha anụnụ anụnụ nke gbanwere na-acha edo edo ka ịgbakwunye ngwọta nkwụsị acidic.Njupụta nke edo edo dabara na ọnụọgụ dsRNA echere na echere na efere.A na-atụ ihe nnabata ahụ na 450 nm.

Ngwa

Ngwa a bụ maka nha nha nke dsRNA fọdụrụnụ.

Ngwa ngwa

Nchekwa na kwụsie ike

1. N'ihi na-ejighi ngwa: The dum kit nwere ike ịchekwa na 2 ~ 8 ℃ na shelf ndụ.Ekwesịrị izere ọkụ siri ike maka nkwụsi ike nchekwa.

2. Maka ngwa eji eme ihe: Ozugbo e meghere microplate, biko kpuchie olulu mmiri ndị na-ejighị ya na efere efere ma laghachi na mpempe akwụkwọ foil nke nwere mkpọ desiccant, zip-achie akpa foil wee laghachi na 2 ~ 8 ℃ ozugbo enwere ike mgbe ejiri ya.Ndị ọzọ reagents kwesịrị ịlaghachi na 2 ~ 8 ℃ ozugbo enwere ike mgbe ejiri ya.

Ihe achọrọ mana anaghị enye ya

1. Microplate agụ na 450 ± 10nm nyo (mma ma ọ bụrụ na ike ịchọpụta na 450 na 650 nm wavelength).

2. Microplate shaker.

3. Atụmatụ na-enweghị RNase na tubes centrifuge.

Chart eji arụ ọrụ

Tupu Ị Malite

1. Weta ihe niile kit components na samples na ụlọ okpomọkụ (18-25 ℃) tupu eji.Ọ bụrụ na agaghị eji ngwa ahụ n'otu oge, biko wepụta naanị ibe na reagents maka nnwale ugbu a, ma hapụ ibe na reagents ndị fọdụrụ n'ọnọdụ achọrọ.

2. Nkịta na-asachapụ: jiri 760mL nke deionized ma ọ bụ mmiri a na-agbanye mmiri mee ka 40mL nke 20 × na-asachapụ ihe na-edozi ahụ iji kwadebe 800mL nke ihe nchekwa 1 ×.

3. Standard: nwa oge tụgharịa ma ọ bụ centrifuge ngwaahịa ngwọta tupu eji.Ntinye nke ụkpụrụ anọ enyere bụ 5ng/μL.Maka ụkpụrụ UTP na pUTP dsRNA, biko gbagharịa ngwa ahịa ngwaahịa ka ọ bụrụ 1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL na ihe nchekwa STE iji see usoro ọkọlọtọ.Maka ụkpụrụ N1-Me-pUTP dsRNA, biko gbagharịa ngwa ahịa ngwaahịa ka ọ bụrụ 2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL na ihe nchekwa STE iji see usoro ọkọlọtọ.Maka ọkọlọtọ 5-OMe-UTP dsRNA, biko tụgharịa ngwaahịa ngwaahịa ahụ ka ọ bụrụ 4,2,1,0.5, 0.25,0.125,0.0625, 0pg/μL na ihe nchekwa STE iji see usoro ọkọlọtọ.Anyị na-akwado na enwere ike ịgbaze ụkpụrụ dị ka eserese ndị a:

4. Biotinylated nchọpụta mgbochi mgbochi na HRP-streptavidin na-arụ ọrụ ngwọta: obere tụgharịa ma ọ bụ centrifuge ngwaahịa ngwọta tupu ojiji.Gwakọta ha na ntinye uche na-arụ ọrụ na ihe nchekwa dilution.

5. TMB substate: aspirate mkpa onunu ogwu nke ngwọta na igba ogwu ndụmọdụ na atụfukwala ihe fọdụrụnụ ngwọta n'ime vial ọzọ.Substate TMB na-enwe mmetụta maka ọkụ, ekpuchila mkpụrụ osisi TMB na ìhè ruo ogologo oge.

Iji protocol

1. Kpebie ọnụọgụ nke ibe achọrọ maka nyocha.Fanye ibe n'ime okpokolo agba maka ojiji.Ekwesịrị ịkwakọba ibe efere efere ndị fọdụrụ na-adịghị eji na nyocha a n'ime akpa ahụ na-eji desiccant.Mechie akpa ahụ nke ọma maka nchekwa friji.

2. Tinye 100μL nke ọ bụla dilutions nke ọkọlọtọ, oghere na sample n'ime olulu mmiri kwesịrị ekwesị.Na-ekpuchi ihe mkpuchi efere.Tinyegharịa maka awa 1 n'ime ụlọ na ịma jijiji na 500rpm.Ekwesịrị ịgbanye ihe nlele ahụ na ihe nchekwa STE na ntinye uche kwesịrị ekwesị maka nyocha ziri ezi.

3. Na-asa nzọụkwụ: Aspirate ihe ngwọta na-asa na 250μL na-asacha ihe nchekwa na nke ọ bụla nke ọma ka ọ guzo 30s.Tụfuo ihe nkpuchi na-asachapụ kpamkpam site n'itinye efere ahụ n'ime akwụkwọ na-amị amị.Na-asachapụ ugboro anọ.

4. Tinye 100μL nke biotinylated antibody na-arụ ọrụ ngwọta n'ime olulu mmiri ọ bụla.Na-ekpuchi ihe mkpuchi efere.Tinyegharịa maka awa 1 n'ime ụlọ na ịma jijiji na 500rpm.

5. Tinyegharịa nzọụkwụ ịsa ahụ.

6. Tinye 100μL nke HRP-streptavidin na-arụ ọrụ ngwọta n'ime olulu mmiri ọ bụla.Na-ekpuchi ihe mkpuchi efere.Tinyegharịa maka minit 30 n'ime ụlọ okpomọkụ na ịma jijiji na 500rpm.

7. Tinyegharịa nzọụkwụ ịsa ahụ ọzọ.

8. Tinye 100μL nke ihe ngwọta TMB n'ime olulu mmiri ọ bụla.Na-ekpuchi ihe mkpuchi efere.Tinye maka nkeji 30 na RT Chebe site na ọkụ.Mmiri mmiri ga-acha anụnụ anụnụ site na mgbakwunye nke ngwọta mkpụrụ.

9. Tinye 50μL ngwọta nkwụsị n'ime olulu mmiri ọ bụla.Mmiri mmiri ga-agbanwe edo edo site na mgbakwunye nke ngwọta nkwụsị.Wee mee ihe na-agụ microplate ma mee nha na 450nm ozugbo.

Mgbakọ nke nsonaazụ

1. Nkezi agụmagụ oyiri maka ọkọlọtọ ọ bụla, njikwa na nlere anya wee wepụ nkezi ihe njupụta ngwa anya efu.Rụpụta eriri ọkọlọtọ yana nnabata na axis vetikal (Y) yana itinye uche dsRNA n'okirikiri (X) axis.

2. A na-atụ aro ka iji sọftụwia dabara adaba na kọmpụta mee mgbako dịka ọkachamara curve 1.3 ma ọ bụ ELISA Calc n'ụdị dabara adaba nke 5 ma ọ bụ 4 na-abụghị linear.

Arụmọrụ

1. Mmetụta:

njedebe dị ala nke nchọpụta: ≤ 0.001pg/μL (maka UTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (maka 5-OMe-UTP-dsRNA).

Obere oke nke quntitation: 0.0156 pg/μL (maka UTP-, pUTP-dsRNA), 0.0312 pg/μL (maka N1-Me-pUTP-dsRNA), 0.0625 pg/μL (maka 5-OMe-UTP-dsRNA).

2. Nkenke: CV nke Intra-Assay ≤10%, CV nke Inter-Assay ≤10%

3. Iweghachite: 80% ~ 120%

4.Linearity: 0.0156-0.5pg / μL (maka UTP-, pUTP-dsRNA) 0.0312-1pg / μL (maka N1-Me-pUTP dsRNA), 0.0625-1pg / μL (maka 5-OMe-UTP-dsRNA).

Ntụle

1. TMB mmeghachi omume okpomọkụ na oge dị oke egwu, biko jikwaa ha dị ka ntụziaka ahụ siri dị.

2. Iji nweta ezi nyocha reproducibility na uche, ịsacha efere nke ọma iji wepụ ngafe na-enweghị mmeghachi omume reagents dị mkpa.

3. All reagents ga-agwakọta nke ọma tupu ojiji na-ezere bumbles n'oge sample ma ọ bụ reagents mgbakwunye.

4. Ọ bụrụ na kristal etolitela n'ime ihe nchekwa nchekwa ndọpụ (20x), kpoo ọkụ ruo 37 ℃ ma gwakọta nwayọ ruo mgbe agbazere kristal kpamkpam.

5. Zere nyocha nke ihe nlele nwere Sodium Azide (NaN3), n'ihi na ọ nwere ike ibibi ọrụ HRP nke na-ebute enweghị atụ nke ọnụọgụ dsRNA.

6. Zere mmetọ RNase n'oge nyocha.

7. Enwere ike ịme ọkọlọtọ / ihe nlele, mgbochi nchọpụta na SA-HRP na RT na-enweghị ịma jijiji, mana nke a nwere ike ime ka nchọpụta nchọpụta mbelata site na otu okpukpu.Maka nke a, anyị na-akwado ụkpụrụ UTP na pUTP dsRNA ga-agbaze site na 2pg/μL, ụkpụrụ N1-Me-pUTP dsRNA kwesịrị ịdịluted site na 4pg/μL na ọkọlọtọ 5-OMe-UTP dsRNA kwesịrị ịdịluted site na 8pg/μL.Na mgbakwunye, incubate HRP-streptavidin na-arụ ọrụ ngwọta maka 60min na ụlọ okpomọkụ.Ejila flask shaker, n'ihi na shaker flask nwere ike ibute nsonaazụ na-ezighi ezi.

Nsonaazụ a na-ahụkarị

1. Standard curve data

2. Standard curve ngụkọta oge

3. Usoro nchọpụta Liner: 0.0312- 1pg / μL