Extraction of Viral RNA from Surface-Bound Virions Using a Silica Spin Column

Özet

Source: Klaus, J. P. et al. Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment. J. Vis. Exp. (2016)

This video demonstrates the extraction of viral RNA from surface-bound virions on epithelial cells using silica spin columns. The method includes chemical lysis, RNA stabilization, and membrane-based purification for downstream quantification of virus-cell attachment.

Protokol

Viral RNA Extraction

1. Purify viral RNA from Junín virus (JUNV) C#1 particles attached to the monolayers using a commercial kit (e.g., the RNeasy Mini kit) according to the manufacturer's protocol. Specifically, follow the "purification of total RNA from animal cells using spin technology protocol" on pages 23-28 in the RNeasy Mini Handbook (4th Edition, April 2006) with the modifications listed below.
   1. Lyse the cells by adding 350 µl of lysis buffer RLT directly to each well. Mix the buffer several times to ensure cell lysis. Note that cells easily lyse in this buffer, and complete lysis can be verified by viewing wells under an inverted microscope.
   2. Transfer the resulting cell lysate to the kit column and follow the rest of the protocol as described. At this point, the virus has been neutralized by the lysis buffer, so the remaining steps of the protocol can be done outside of the class II biosafety cabinet, provided the kit tubes were not contaminated with infectious virus.
   3. Include the optional step from the manufacturer's protocol, which is an additional centrifugation of the spin column to eliminate any possible carryover of Buffer RPE.
   4. In the final step, the manufacturer's protocol, elute the purified RNA from the spin column using 30 µl of nuclease-free ddH₂O. Store RNA at -80 °C at this point or use directly in the quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. To prevent degradation of purified viral RNA samples by nucleases, use nuclease-free reagents and equipment, and keep thawed RNA samples on ice.
      NOTE: Buffers RLT and RW1 contain guanidine salt and should not be mixed with bleach. Buffer RW1 contains ethanol.

Malzemeler

Bu makalede kullanılan malzemelerin listesi

Ad	Şirket	Katalog numarası	Yorumlar
Vero E6 cells	American Type Culture Collection	CRL-1586
Costar 48-well plate	Corning	3548
RNeasy mini kit	Qiagen	74106	Do not mix buffers RLT or RW1 with bleach
QIAshredder homogenizer	Qiagen	79654
RNase Inhibitor (5000U/100 µl)	Life Technologies	N808-0119