RNA mai madauri biyu (dsRNA) ELISA KIT

Wannan kit ɗin shine Enzyme-Linked Immunosorbent Assay (ELISA) mai haɗawa tare da tsarin biotin-Streptavidin, don ƙididdige ma'aunin dsRNA mai tsayi sama da 60 tushe nau'i-nau'i(bp), ba tare da la'akari da jerin ba.An riga an lulluɓe farantin da anti-dsRNA antibody.Ana ƙara dsRNA da ke cikin samfurin kuma yana ɗaure ga ƙwayoyin rigakafi da aka lulluɓe akan rijiyoyin.Sannan ana ƙara anti-dsRNA antibody biotinylated kuma yana ɗaure zuwa dsRNA a cikin samfurin.Bayan wankewa, ana ƙara HRP-Streptavidin kuma yana ɗaure ga anti-dsRNA antibody Biotinylated.Bayan shiryawa unbound HRP-Streptavidin an wanke shi.Sa'an nan kuma TMB substrate bayani da aka kara da catalyzed ta HRP don samar da wani blue launi samfurin canza zuwa rawaya bayan ƙara acidic tasha bayani.Girman launin rawaya ya yi daidai da adadin dsRNA da aka yi niyya a faranti.Ana auna abin sha a 450 nm.

Aikace-aikace

Wannan kit ɗin don auna ƙididdigewa ne na saura dsRNA.

Abubuwan Kit

Adana da Kwanciyar hankali

1. Don kayan aikin da ba a yi amfani da su ba: Za a iya adana kayan duka a 2 ~ 8 ℃ a rayuwar shiryayye.Ya kamata a guje wa haske mai ƙarfi don kwanciyar hankali.

2. Don kit ɗin da aka yi amfani da shi: Da zarar an buɗe microplate, da fatan za a rufe rijiyoyin da ba a amfani da su tare da madaidaicin farantin karfe kuma ku koma jakar jakar da ke ɗauke da fakitin desiccant, zip-hatimi jakar jakar ku kuma komawa zuwa 2 ~ 8 ℃ da wuri-wuri bayan amfani.Sauran reagents ya kamata a mayar da su zuwa 2 ~ 8 ℃ da wuri-wuri bayan amfani.

Kayayyakin da ake buƙata Amma Ba a Ba su ba

1. Mai karanta Microplate tare da 450 ± 10nm tace (mafi kyau idan za'a iya ganowa a 450 da 650 nm raƙuman ruwa).

2. Microplate shaker.

3. Tukwici marasa RNase da bututun centrifuge.

Taswirar Tafiya

Kafin Ka Fara

1. Kawo duk kayan aikin kit da samfurori zuwa zafin jiki (18-25 ℃) kafin amfani.Idan ba za a yi amfani da kit ɗin cikin lokaci ɗaya ba, da fatan za a fitar da tsiri da reagents don gwajin na yanzu, kuma a bar sauran tsiri da reagents cikin yanayin da ake buƙata.

2. Wanke buffer: tsarma 40mL na 20 × mai da hankali mai buffer tare da 760mL na deionized ko distilled ruwa don shirya 800mL na 1 × wanke buffer.

3. Daidaito: a taƙaice juya ko saka hannun jari kafin amfani.Matsakaicin ma'auni huɗu da aka bayar shine 5ng/μL.Don ma'auni na UTP da pUTP dsRNA, da fatan za a tsarma maganin hannun jari zuwa 1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL tare da buffer STE don zana daidaitaccen lanƙwasa.Don ma'aunin N1-Me-pUTP dsRNA, da fatan za a tsarma maganin haja zuwa 2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL tare da buffer STE don zana daidaitaccen lanƙwasa.Don ma'aunin 5-OMe-UTP dsRNA, da fatan za a tsarma maganin hannun jari zuwa 4,2,1,0.5, 0.25,0.125,0.0625, 0pg/μL tare da buffer STE don zana daidaitaccen lanƙwasa.Muna ba da shawarar za a iya diluted ƙa'idodi kamar yadda ginshiƙi masu zuwa:

4. Biotinylated gano antibody da HRP-streptavidin aiki bayani: a taƙaice juya ko centrifuge da stock bayani kafin amfani.Tsarma su zuwa aikin maida hankali tare da buffer dilution.

5. TMB substate: aspirate da ake bukata sashi na maganin tare da haifuwa tukwici kuma kada a sake jujjuya ragowar maganin a cikin vial.Ƙarƙashin ƙasa na TMB yana kula da haske, kar a watsa TMB substrate zuwa haske na dogon lokaci.

Amfani da yarjejeniya

1. Ƙayyade adadin adadin da ake buƙata don tantancewa.Saka firam ɗin a cikin firam don amfani.Ragowar filayen farantin da ba a yi amfani da su ba a cikin wannan gwajin ya kamata a sake tattara su a cikin jaka tare da na'urar bushewa.Rufe jakar sosai don ajiyar firiji.

2. Ƙara 100μL kowane dilutions na daidaitattun, blank da samfurori a cikin rijiyoyin da suka dace.Rufe da farantin karfe.Sanya na tsawon 1hr a zafin jiki tare da girgiza a 500rpm.Ya kamata a narke samfuran tare da buffer STE zuwa maida hankali mai dacewa don ingantaccen ƙima.

3. Wanke mataki: aspirate maganin kuma wanke tare da 250μL wash buffer zuwa kowace rijiya a bar shi ya tsaya 30s.Yi watsi da buffer ɗin gaba ɗaya ta hanyar ɗora farantin a kan takarda mai sha.Gaba ɗaya wanke sau 4.

4. Add 100μL na biotinylated gano antibody aiki bayani a cikin kowace rijiya.Rufe da farantin karfe.Sanya na tsawon 1hr a zafin jiki tare da girgiza a 500rpm.

5. Maimaita matakin wankewa.

6. Ƙara 100μL na HRP-streptavidin aiki bayani a cikin kowace rijiya.Rufe da farantin karfe.Sanya na tsawon minti 30 a zafin jiki tare da girgiza a 500rpm.

7. Maimaita matakin wankewa kuma.

8. Add 100μL na TMB substrate bayani a kowace rijiya.Rufe da farantin karfe.Incubate na minti 30 a RT Kariya daga haske.Ruwan zai zama shuɗi ta hanyar ƙara bayani na substrate.

9. Ƙara 50μL na maganin tsayawa a kowace rijiya.Ruwan zai juya rawaya ta hanyar ƙara maganin tasha.Sa'an nan kuma kunna microplate reader kuma gudanar da ma'auni a 450nm nan da nan.

Lissafin Sakamako

1. Matsakaicin kwafin karatun ga kowane ma'auni, sarrafawa, da samfurori kuma cire matsakaicin matsakaicin sifilin ma'aunin gani.Gina madaidaicin madaidaicin lanƙwasa tare da ɗaukar hankali akan axis (Y) a tsaye da tattarawar dsRNA akan a kwance (X) axis.

2. Ana ba da shawarar yin lissafin tare da software mai dacewa da tsarin kwamfuta kamar ƙwararren ƙwararren 1.3 ko ELISA Calc a cikin sigina 5 ko 4 wanda ba daidai ba.

Ayyuka

1. Hankali:

ƙananan iyaka na ganowa: ≤ 0.001pg/μL (na UTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (na 5-OMe-UTP-dsRNA).

ƙananan iyaka na ƙididdigewa: 0.0156 pg/μL (na UTP-, pUTP-dsRNA), 0.0312 pg/μL (na N1-Me-pUTP-dsRNA), 0.0625 pg/μL (na 5-OMe-UTP-dsRNA).

2. Daidaitawa: CV na Intra-Assay ≤10%, CV na Inter-Assay ≤10%

3. Farfadowa: 80% ~ 120%

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

La'akari

1. TMB zazzabi dauki da lokaci yana da mahimmanci, da fatan za a sarrafa su bisa ga umarnin sosai.

2. Domin cimma mai kyau assay reproducibility da ji na ƙwarai, dace wanke faranti don cire wuce haddi un-reacted reagents yana da muhimmanci.

3. Duk da reagents ya kamata a gauraye sosai kafin amfani da kuma kauce wa bumbles a lokacin samfurin ko reagents Bugu da kari.

4. Idan lu'ulu'u sun samo asali a cikin ma'aunin wanke-wanke mai da hankali (20x), dumi zuwa 37 ℃ da haɗuwa a hankali har sai lu'ulu'u sun narkar da su gaba daya.

5. A guji tantance samfuran da ke ɗauke da Sodium Azide (NaN3), saboda zai iya lalata ayyukan HRP wanda ke haifar da ƙarancin ƙima na adadin dsRNA.

6. Guji gurɓatar RNase yayin tantancewa.

7. Hakanan ana iya gudanar da ma'auni/samfurin, rigakafin ganowa da SA-HRP a RT ba tare da girgiza ba, amma wannan na iya haifar da raguwar ganewa ta hanyar sau ɗaya.Don wannan yanayin, muna ba da shawarar ka'idodin UTP da pUTP dsRNA yakamata a diluted daga 2pg/μL, N1-Me-pUTP dsRNA daidaitattun yakamata a diluted daga 4pg/μL da 5-OMe-UTP dsRNA daidaitattun yakamata a diluted daga 8pg/μL.Bugu da kari, incubate HRP-streptavidin aiki bayani na 60min a dakin zafin jiki.Kar a yi amfani da abin shaker flask, saboda shaker flask na iya haifar da rashin daidaitaccen sakamako.

Sakamakon na al'ada

1. Daidaitaccen bayanan lankwasa

2. Daidaitaccen lissafin lankwasa

3. Kewayon ganowa na layi: 0.0312- 1pg / μL