IZON RXT01 qEV RNA Extraction Kit Buku Logwiritsa Ntchito

qEV RNA EXTRACTION KIT (RXT01)
MANERO OBUKA

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MFUNDO NDI MALO OGWIRITSIRA NTCHITO PA qEV RNA EXTRACTION KIT (RXT01)
KUCHULUKA KWAMBIRI NDI WOYANG'ANIRA KWA EXTRACELLULAR VESICLE RNA

MISONKHANO YOTANTHAUZIRA NDI KULEMBA

Onetsetsani kuti mukutsatira mfundo zodzitetezera zomwe zaperekedwa mu bukhuli. Chitetezo ndi zidziwitso zina zapadera zidzawonekera m'mabokosi ndikuphatikiza zizindikiro zomwe zili pansipa.
Gulu 1:Chitetezo ndi Zizindikiro Zowopsa

	Chizindikirochi chikuwonetsa upangiri wanthawi zonse wamomwe mungasinthire njira kapena kupangira njira zomwe mungatsatire pazochitika zinazake.
	Chizindikirochi chimasonyeza kumene tiyenera kusamala kwambiri.

Gulu 2: Mawu Ogwiritsidwa Ntchito M'bukuli

TERM	CHIFUKWA
RNA	Ribonucleic acid
EV	Ma vesicles a extracellular
Gawo la qEV Kudzipatula	Mizati yopatula kukula kwa Izon imapatula ma EV kumadzi osiyanasiyana akamadutsa pagawo lodzaza ndi porous. polysaccharide utomoni

MAU OYAMBA

2.1 Kugwiritsa Ntchito
The qEV RNA Extraction Kit ingagwiritsidwe ntchito pochotsa RNA kuchokera ku EVs zomwe zasiyanitsidwa pogwiritsa ntchito mizati ya qEV Kudzipatula ndikukhazikika (ngati kuli kofunikira). The qEV RNA Extraction Kit imathandizira kuchotsa RNA kuchokera ku EVs olekanitsidwa ndi biofluids komanso kuchokera ku cell culture media. M'zigawozo ndi zachindunji, osati kusokonezedwa ndi kukondera kwa kukula, ndipo zimapereka sampkuyeretsedwa kwakukulu komwe kumatha kusinthidwa kukhala ma voliyumu a 50-100 µL (monga momwe wogwiritsa ntchito afotokozera). RNA yoyeretsedwa ndiyoyenera kugwiritsidwa ntchito pamayendedwe akumunsi kuphatikiza qPCR, ma microarrays, ndi ma RNA sequencing (RNA-seq) pazifukwa kuphatikiza kuyang'anira momwe matenda akupitira komanso mayankho achire.
2.2 Kuwongolera Ubwino
Mogwirizana ndi Izon's ISO 13485-certified Quality Management System, gulu lililonse la qEV RNA Extraction Kits limayesedwa motsutsana ndi zomwe zidakonzedweratu kuti zitsimikizire kuti zinthu sizingafanane.
2.3 Zochepa Zogwiritsa Ntchito Zogulitsa
The qEV RNA Extraction Kit idapangidwa kuti izingofufuza zokha. Sichigwiritsidwe ntchito ndi anthu kapena matenda.

MALANGIZO OGWIRITSA NTCHITO MANUKO

Gawo lotsatirali likupereka malangizo ogwiritsira ntchito RXT01 pamanja. Kuti mugwiritse ntchito mizati ya qEV ndi AFC, chonde onani Buku lathunthu la AFC pa
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3.1 Kugwira ntchito ndi RNA
Ma RNase ndi ma enzyme okhazikika komanso amphamvu omwe amawononga RNA yomwe nthawi zonse samachotsedwa mokwanira ndi mayankho a autoclaving ndi glassware. Musanayese kuchotsa RNA kuchokera ku EV sampLes, malo opanda RNase ayenera kupangidwa. Njira zodzitetezera zotsatirazi ndizomwe zimalimbikitsidwa ngati chitetezo chanu chabwino kwambiri pama enzyme awa.

• Dera la RNA liyenera kukhala kutali ndi malo ogwirira ntchito a microbiological.
• Magolovesi aukhondo otayidwa ayenera kuvala nthawi zonse. Magolovesi ayenera kusinthidwa pafupipafupi kuti apewe kuipitsidwa.
• Mayankho osankhidwa, maupangiri, machubu, malaya a labu, ma pipette, ndi zina zotere ziyenera kugwiritsidwa ntchito pazoyeserera zonse zokhudzana ndi RNA. Pulasitiki woperekedwa ndi wogwiritsa ntchito (monga machubu 1.5, 2.0, 15, 50 mL kapena nsonga za pipette) ziyenera kukhala zovomerezeka za RNase/DNase-free.
• Mayankho onse a RNA ayenera kukonzedwa pogwiritsa ntchito osachepera 0.05% DEPCtreated autoclaved water or molecular biology grade RNase/DNase-free water.
• Malo onse ayenera kutsukidwa ndi njira zogulitsira za RNase zochotsa madzi musanayambe kuyesa.
• RNA yoyeretsedwa iyenera kusamaliridwa ndikusungidwa pa ayezi musanagwiritse ntchito komanso panthawi yakutsika.

3.2 Zigawo za Kit
Zigawo za qEV RNA Extraction Kit (50 preps)

zigawo	RXT01 (50 zokonzekera)
Lysis Buffer A	2x30ml pa
Lysis Additive B	7 mL
Wash Solution A	38 mL
Elution Solution A	6 mL
Mipikisano ya Mini Spin	50
Machubu Osonkhanitsa	50
Machubu owonjezera (1.7 mL)	50

Lysis Buffer A ili ndi guanidine thiocyanate ndipo iyenera kusamaliridwa bwino. Guanidine thiocyanate amapanga mankhwala otakasuka kwambiri akaphatikizidwa ndi bulichi, motero kusamala kuyenera kutengedwa kuti muchotse bwino chilichonse mwa njira iyi. Ngati madzi okhala ndi zotchingira izi atayika, yeretsani ndi zotsukira za mu labotale ndi madzi. Ngati madzi otayira ali ndi zinthu zomwe zitha kupatsirana, yeretsani malo omwe akhudzidwawo poyamba ndi zotsukira mu labotale ndi madzi, kenako ndi 1% (v/v) sodium hypochlorite.
Zopangira Makasitomala ndi Zida

• Magolovesi opanda ufa otayidwa
• Benchtop microcentrifuge
• Ma Micropipettes
• Zosefera wosabala ndi pipette
• Vortex
• 96% -100% Molecular Biology Kalasi Ethanol

ZOKHUDZA

Protocol iyi ikufotokoza njira yochotsera RNA kuchokera ku ma EV oyeretsedwa pogwiritsa ntchito mizati ya qEV Isolation.
Musanagwiritse ntchito, chonde onetsetsani izi:

• EV sampLes zasungidwa pa -80 ° C musanagwiritse ntchito (kulephera kutero kudzakhudza zokolola za RNA).
• Onetsetsani kuti njira zonse zili m'chipinda chozizira komanso kuti Lysis Buffer A yatenthedwa kufika 60 ° C kwa mphindi 20 musanagwiritse ntchito. Ngati ma precipitates alipo, sakanizani zonse bwino mpaka zothetsera zimveke bwino.
• Konzani mosakayika wa Wash Solution A powonjezera 90 mL wa 96% -100% ethanol mu botolo lomwe mwapatsidwa (lomwe lili ndi 38 ml ya concentrated Wash Solution A) kuti mupereke voliyumu yomaliza ya 128 mL.
• Masitepe onse a centrifugation amachitidwa kutentha m'machubu oyenera.
• Zozungulira zomwe zaperekedwa zimakonzedwa kuti zigwiritsidwe ntchito ndi ma benchtop centrifuges ndipo sizingagwiritsidwe ntchito ndi vacuum zida.
• Kuyika centrifuging mini spin mizati pa liwiro lokwera kuposa momwe akulimbikitsira kungakhudze zokolola za RNA. Centrifuging pa liwiro otsika kuposa analimbikitsa sizingakhudze RNA zokolola koma kuonjezera elution nthawi.
• Ngati yankho lililonse silidutsa pamizere yozungulira mkati mwa nthawi yodziwika ya centrifugation, zungulirani kwa mphindi zina 1-2 mpaka yankho litadutsa pamzatiwo.
  Osa kupitilira liwiro la centrifugation.

Ndondomeko:

1. Onjezani 900 µL wa Lysis Buffer A ndi 125 µL wa Lysis Additive B mpaka 600 µL EV sampndi kudzipatula pogwiritsa ntchito mizati ya qEV Kudzipatula ndikukhazikika ngati kuli kofunikira.
2. Sakanizani njira ya EV ndi Lysis Buffer/Additive povota kwa masekondi 10 kenaka muyimire pa firiji kwa mphindi 20.
   Zindikirani: Ngati mukugwiritsa ntchito qEV Concentration Kit, patulani tinthu tating'ono ta Nanotrap® kuchokera ku sample mwa centrifuging the particle-sampLe kuyimitsidwa pa 16,800 RCF kwa mphindi 10 kutentha firiji. Popanda kusokoneza Nanotrap® EV tinthu tating'onoting'ono pellet, sinthani sample supernatant mu chubu choyera.
3. Onjezani 1.5 mL wa 96% -100% ethanol ku osakaniza kuchokera Gawo 2 ndikusakaniza bwino ndi vortexing kwa masekondi 10.
4. Chotsani 700 µL ya zosakaniza kuchokera ku Gawo 3 kukhala gawo laling'ono lozungulira. Centrifuge mu benchi pamwamba centrifuge kwa mphindi 1 pa 3,300 × g.
   Tayani zomwe zikuyenda ndikuphatikizanso gawo laling'ono lozungulira ndi chubu chake.
5. Bwerezani Gawo 4 nthawi zambiri ngati kuli kofunikira kusamutsa kusakaniza kotsalira kuchokera ku Gawo 3 kupita ku gawo laling'ono lozungulira ndikutaya kutuluka.
6. Ikani 600 µL ya Wash Solution A pazanja ndi centrifuge mu bench-top centrifuge kwa masekondi 30 pa 3,300 × g. Tayani zotulukapo ndikuphatikizanso ndime yozungulira ndi chubu chake chosonkhanitsira.
7. Bwerezani Gawo 6 kamodzinso ndikutaya njirayo.
8. Sinthani ndime yopanda kanthu kwa mphindi imodzi pa 1 × g kuti muchotse madzi otsala. Tayani chubu chotolera.
9. Tumizani gawo lozungulira ku chubu chatsopano cha 1.7mL elution. Ikani 50 µL ya Elution Solution A pazanja ndi centrifuge kwa mphindi imodzi pa 1 × g, ndikutsatiridwa ndi mphindi zina 400 pa 2 × g.
10. Kuti muchiritse kwambiri, tumizani yankho lochotsedwa ku gawolo ndikusiya gawolo liyime pa kutentha kwapakati kwa mphindi ziwiri. Centrifuge kwa mphindi imodzi pa 2 × g, ndikutsatiridwa ndi mphindi 1 pa 400 × g.

MAFUNSO AMENE AMAFUNSA KAWIRIKAWIRI (FAQS)

Kodi chimachitika ndi chiyani ngati centrifuge yosinthika sikupezeka?
Centrifuge yokhazikika imatha kugwiritsidwa ntchito malinga ngati liwiro silikupatula malingaliro athu; komabe, zokolola zochepa zimatha kuwonedwa.
Kodi chingachitike ndi chiyani ngati ndipanga centrifugation pa kutentha kolakwika?
Mpofunika kuchita masitepe onse centrifugation kutentha firiji; Komabe, centrifugation pa 4 ° C sikudzasokoneza magwiridwe antchito a zida.
Kodi chimachitika ndi chiyani ndikawonjezera ma reagents ochulukirapo?
Izi zitha kuchepetsa mtundu ndi kuchuluka kwa RNA yoyeretsedwa.
Kukweza RNA yanu m'mavoliyumu apamwamba kumawonjezera zokolola koma zotsatira zakeampLe adzakhala ndi ndende yochepa ya RNA. Eluting m'mavoliyumu ang'onoang'ono adzawonjezera ndende koma kuchepetsa zokolola zonse.
Kodi chimachitika ndi chiyani ngati ndiiwala kupota mowuma ndisanathe gawo langa lomaliza?
RNA yoyeretsedwa idzaipitsidwa ndi Sambani Njira A yomwe ingachepetse ubwino wa RNA yoyeretsedwa ndikusokoneza ntchito zapansi.
Kodi ndingathe kubwerezanso kachiwiri?
Inde, koma tikulimbikitsidwa kuti kufotokozera kwachiwiri kukhale mu voliyumu yaying'ono (monga 50% ya kutulutsa koyamba). Ndi bwino kuchita yachiwiri elution mu osiyana elution chubu kupewa diluting woyamba elution.
Chifukwa chiyani wangaamples kusonyeza zokolola zochepa za RNA?
Kuchuluka kwa RNA komwe kuli mkati mwa ma EV ndi kochepa kwambiri ndipo kumatha kusiyanasiyana kutengera ma sample kuti anali olekanitsidwa ndi njira zoyeretsera. Zokolola za RNA zitha kuchulukitsidwa powonjezera kuchuluka kwa sample input; komabe, kuti mupeze zotsatira zabwino komanso zokhazikika qEV RNA Extraction Kit iyenera kugwiritsidwa ntchito limodzi ndi qEV Isolation columns ndi qEV Concentration Kit.
Chifukwa chiyani chiŵerengero cha A260/280 cha RNA yoyeretsedwa chili chotsika kuposa 2.0?
Zambiri za RNA zotengedwa ku EVs zidzakhala zidutswa zazifupi za RNA zomwe zimakhala zotsika kwambiri. Chiŵerengero cha A260/280 chimakonda kuchepa ndi kuchepa kwa RNA; ndizabwinobwino kupeza chiyerekezo cha A260/280 cha 1–1.6 pamiyezo yanu yoyeretsedwaample. Chiŵerengero chotsika cha A260/280 sichingakhudze ntchito iliyonse yotsika.
Chifukwa chiyani RNA yanga yochotsedwa sichita bwino kunsi kwa mtsinje mapulogalamu?
Ngati chotchinga chotchinga china chosiyana ndi chomwe chidaperekedwa mu zidacho chidagwiritsidwa ntchito, chikhoza kusokoneza kutsika kwapansi. Zigawo zodziwika bwino zomwe zimadziwika kuti zimasokoneza ndi kuchuluka kwa mchere (kuphatikiza EDTA), zotsukira, ndi ma denaturing agents. Yang'anani kugwirizana kwa bafa yanu ya elution ndi ntchito yomwe mukufuna.

ZOTHANDIZA ZINA

Onani ku Satifiketi Yachitetezo kuti mugawike ndikuyikapo zoopsa komanso mawu osamala.
The Safety Data Sheet ya qEV RNA Extraction Kit ili pa www.izon.com/products/sds
Onani Migwirizano ndi Migwirizano pa www.izon.com/wp-content/uploads/Terms-and-Conditions.pdf

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Zolemba / Zothandizira

IZON RXT01 qEV RNA Extraction Kit [pdf] Buku Logwiritsa Ntchito RXT01, qEV RNA Extraction Kit

Zothandizira

• Dzipatulani & Muyese Zowonjezera Zowonjezera & Nanoparticles
• Izon Science Safety Data Sheets (SDS)