RNA fa'alua (dsRNA) ELISA KIT

O lenei pusa o le Enzyme-Linked Immunosorbent Assay (ELISA) faʻatasi ma le biotin-Streptavidin system, mo le fua faʻatatau o le dsRNA ma le umi i luga ole 60 base pairs(bp), e tusa lava po o le a le faʻasologa.O le ipu ua uma ona ufiufi i le anti-dsRNA antibody.dsRNA o loʻo iai i le faʻataʻitaʻiga ua faʻaopoopoina ma fusifusia i antibodies o loʻo ufiufi i luga o vaieli.Ona faaopoopo lea o le anti-dsRNA antibody biotinylated ma fusifusia i le dsRNA i le faʻataʻitaʻiga.A maeʻa ona fufulu, faʻaopoopo HRP-Streptavidin ma fusifusia i le antibody anti-dsRNA Biotinylated.A mae'a ona fa'atosina ona fufulu ese lea o le HRP-Streptavidin.Ona fa'aopoopoina lea o le vaifofo o le substrate TMB ma fa'apipi'iina e le HRP e maua ai se oloa lanu lanumoana na suia i le samasama pe a uma ona fa'aopoopo le vaifofo fa'agata.Ole tele ole samasama e fa'atatau ile aofa'i ole dsRNA na pu'eina ile ipu.O le absorbance e fuaina ile 450 nm.

Fa'aaogāga

O lenei pusa e mo le fua fa'atatau o le dsRNA totoe.

Vaega o pusa

Teuina ma le mautu

1. Mo pusa e le faʻaaogaina: O le pusa atoa e mafai ona teuina i le 2 ~ 8 ℃ i le fata ola.E tatau ona aloese mai le malamalama malosi mo le mautu o le teuina.

2. Mo pusa faʻaoga: A maeʻa loa le microplate, faʻamolemole ufiufi vaieli e leʻi faʻaaogaina i le faʻamaufaʻailoga ipu ma toe foʻi i le taga pepa o loʻo i ai le pusa faʻamaʻi, faʻamaufaʻailoga le pusa pepa ma toe foʻi i le 2 ~ 8 ℃ i se taimi vave pe a uma ona faʻaoga.O isi reagents e tatau ona toe faʻafoʻi i le 2 ~ 8 ℃ i se taimi vave pe a uma ona faʻaoga.

Mea e Manaomia Ae Le Tuuina Atu

1. Microplate faitau ma 450±10nm faamama (sili atu pe a mafai ona iloa i le 450 ma 650 nm wavelength).

2. Lu'u fa'ameamea.

3. RNase-saoloto fautuaga ma centrifuge paipa.

Fa'agaioiga Fa'asologa

Ae E te Le'i Amata

1. Aumai vaega uma o pusa ma fa'ata'ita'iga ile vevela potu (18-25℃) a'o le'i fa'aoga.Afai e le fa'aaogaina le pusa i le taimi e tasi, fa'amolemole na'o le ave i fafo o fasipepa ma mea fa'afofoga mo le fa'ata'ita'iga o lo'o iai nei, ae tu'u fasi fasi pepa ma reagents o totoe ile tulaga mana'omia.

2. Fufulu fa'apala: fa'asusu le 40mL o le 20x fa'apolopolo fufulu fa'atasi ma le 760mL o le vai fa'amuta po'o le vai fa'amama e saunia ai le 800mL o le 1x fa'amama fufulu.

3. Tulaga masani: vili pu'upu'u pe centrifuge le vaifofo fa'asoa a'o le'i fa'aogaina.O le faʻatonuga o faʻataʻitaʻiga o loʻo tuʻuina atu o le 5ng / μL.Mo tulaga UTP ma le pUTP dsRNA, fa'amolemole fa'amalie le vaifofo fa'asoa i le 1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL ma le STE buffer e tusi ai le pi'o masani.Mo tulaga N1-Me-pUTP dsRNA, fa'amolemole fa'amalie le vaifofo fa'asoa i le 2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL fa'atasi ma le STE buffer e tusi ai le pi'o masani.Mo le tulaga 5-OMe-UTP dsRNA, fa'amolemole fa'amalie le vaifofo fa'asoa i le 4,2,1,0.5, 0.25,0.125,0.0625, 0pg/μL fa'atasi ma le STE buffer e tusi ai le pi'o masani.Matou te fautuaina tulaga e mafai ona faʻafefe e pei o siata nei:

4. Biotinylated detection antibody ma HRP-streptavidin fofo galue: vili puupuu pe centrifuge le vaifofo faʻasoa aʻo leʻi faʻaaogaina.Fa'asusu i latou i le fa'atonuga galue ma le fa'afefete fa'afefeteina.

5. TMB substate: fa'asusu le vaila'au mana'omia o le vaifofo fa'atasi ai ma fautuaga fa'amama ma 'aua ne'i toe lafo le vaifofo o totoe i totonu o le fagu.TMB substate e maaleale i le malamalama, aua le faʻaalia le TMB substrate i le malamalama mo se taimi umi.

Fa'aaogaina o le protocol

1. Fuafua le numera o fasi pepa e mana'omia mo le su'ega.Fa'aofi fasipepa i totonu o fa'avaa mo le fa'aoga.O fasi fasi ipu e totoe e le'o fa'aaogaina i lenei su'ega e tatau ona toe teu i totonu o le ato ma fa'amago.Tapuni lelei le ato mo le teuina i totonu o le pusaaisa.

2. Faʻaopoopo 100μL taʻitasi faʻafefeteina o tulaga masani, avanoa ma faʻataʻitaʻiga i totonu o vaieli talafeagai.Ufiufi i le ipu fa'amau.Taofi mo le 1 itula ile vevela ole potu ma lulu ile 500rpm.O fa'ata'ita'iga e tatau ona fa'afefeteina ile STE pa'u ile fa'atonuga talafeagai mo su'ega sa'o.

3. Laasaga Fufulu: Fa'asusu le vaifofo ma fufulu ile 250μL fufulu fa'amau i vaieli ta'itasi ma tu'u ai mo le 30s.Lafoa'i atoa le pa'u fufulu e ala i le tu'iina o le ipu i luga o le pepa fa'afefe.Fufulu atoa 4 taimi.

4. Fa'aopoopo le 100μL ole su'esu'ega biotinylated vaifofo galue ile vaieli ta'itasi.Ufiufi i le ipu fa'amau.Taofi mo le 1 itula ile vevela ole potu ma lulu ile 500rpm.

5. Toe fai le la'asaga fufulu.

6. Faʻaopoopo 100μL o HRP-streptavidin fofo galue i totonu o vaieli taitasi.Ufiufi i le ipu fa'amau.Taofi mo le 30 minute ile vevela ole potu ma lulu ile 500rpm.

7. Toe fai le la'asaga fufulu.

8. Faaopopo le 100μL o le TMB substrate solution i totonu o vaieli taitasi.Ufiufi i le ipu fa'amau.Taofi mo le 30 min ile RT Puipui mai le malamalama.O le a liu lanumoana le vai e ala i le faʻaopoopoina o se vaifofo substrate.

9. Faaopopo le 50μL o le vaifofo taofi i totonu o vaieli taitasi.Ole vai ole a liu samasama ile fa'aopoopoina ole fofo taofi.Ona fa'asolo lea o le microplate faitau ma fa'atino fua ile 450nm vave.

Fuafuaga o I'uga

1. Fa'atauva'a faitauga fa'alua mo ta'iala ta'itasi, fa'atonutonu, ma fa'ata'ita'iga ma to'ese le averesi fa'ata'ita'i fa'ata'ita'i e leai se maualuga.Fausia se pi'o fa'ata'ita'i fa'atasi ai ma le fa'amago i luga o le tu'usa'o (Y) axis ma le fa'atonuga o le dsRNA i luga o le laina fa'ata'atia(X).

2. E fautuaina le faia o le faʻatusatusaga i komepiuta faʻapipiʻi faʻapipiʻi faʻapipiʻi komepiuta e pei o le curve expert 1.3 poʻo le ELISA Calc i le 5 poʻo le 4 faʻataʻitaʻiga e le faʻaogaina laina.

Fa'atinoga

1. Ma'ale'ale:

tapulaa pito i lalo ole iloa: ≤ 0.001pg/μL (mo UTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (mo 5-OMe-UTP-dsRNA).

pito i lalo ole quantitation:0.0156 pg/μL (mo UTP-, pUTP-dsRNA),0.0312 pg/μL (mo N1-Me-pUTP-dsRNA),0.0625 pg/μL (mo 5-OMe-UTP-dsRNA).

2. Sa'o: CV o Intra-Assay ≤10%, CV o Inter-Assay ≤10%

3. Toe faʻaleleia: 80% ~ 120%

4.Linearity:0.0156-0.5pg/μL(moUTP-,pUTP-dsRNA)0.0312-1pg/μL(moN1-Me-pUTP dsRNA),0.0625-1pg/μL(mo 5-OMe-UTP-dsRNA).

Manatuga

1. TMB tali vevela ma le taimi e taua, faamolemole pulea i latou e tusa ai ma faatonuga strictly.

2. Ina ia ausia le toe gaosia lelei ma le maaleale, o le fufuluina tatau o papatusi e aveese ai le tele o mea e le tali atu e taua.

3. E tatau ona fa'afefiloi mae'ae'a uma mea fa'afoliga a'o le'i fa'aogaina ma 'alofia fa'alavelave i le taimi o fa'ata'ita'iga po'o le fa'aopoopoina o reagents.

4. Afai ua fa'atupu tioata i totonu o le pa'u fufulu fa'ato'a (20x), fa'amafanafana i le 37 ℃ ma fa'afefiloi malie se'ia mae'a fa'amavae atoa tioata.

5. Aloese mai le su'esu'eina o fa'ata'ita'iga o lo'o i ai le Sodium Azide (NaN3), aua e mafai ona fa'aleagaina ai le gaioiga a le HRP e mafua ai ona le fa'atatauina le aofa'i o le dsRNA.

6. Aloese mai le fa'aleagaina o le RNase ile taimi ole su'ega.

7. O le tulaga masani/fa'ata'ita'iga, su'esu'ega antibody ma le SA-HRP e mafai fo'i ona fa'atinoina ile RT e aunoa ma le luluina, ae e ono fa'aitiitia ai le lagona fa'aalia ile fa'a-tasi.Mo lenei tulaga, matou te fautuaina UTP ma pUTP dsRNA tulaga faatonuina e tatau ona diluted mai 2pg / μL, N1-Me-pUTP dsRNA tulaga faatonuina e tatau ona diluted mai 4pg / μL ma 5-OMe-UTP dsRNA tulaga faatonuina e tatau ona diluted mai 8pg / μL.E le gata i lea, faʻapipiʻi le HRP-streptavidin fofo galue mo le 60min i le vevela o le potu.Aua le fa'aogaina le fagu lulu, aua e le sa'o le i'uga o le fagu lulu.

I'uga masani

1. Faʻamaumauga faʻasologa masani

2. Fa'atatauga pi'o masani

3. Laina va'aiga iloa:0.0312- 1pg/μL