Purification of influenza A virus RNA from cell supernatant to check sequences
Marta Gaglia, lea.gaucherand
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Abstract
We use this protocol to isolate RNA from influenza A virus-containing cell supernatant and monitor maintenance of sequence insertions or mutations
Steps
Influenza A virus extraction from cell supernatant using QIAamp Viral RNA Mini Kit
Follow the manufacturer's protocol using a starting cell supernatant volume of 140 µl.
Elute in 60 µl buffer AVE.
DNase treatment using Qiagen RNase-free DNase set:
Make mastermix of DNase and buffer. For each sample:
- 10 µl buffer RDD
- 2.5 µl DNase I Add to each RNA tube, 27.5 µl H2O , and 12.5 µl DNase master mix.
Incubate at RT for 10min.
Purification of RNA using Zymo RNA clean up and concentrator kit:
Follow the manufacturer's protocol, adding 100 µl RNA binding buffer and 300 µl ethanol as the initial step before transferring to column
Elute in 15 µl water.
Note: Can also use RNeasy MinElute kit, using manufacturer's protocol
Reverse transcription with iScript.
For each sample, mix:
- 1 µl purified RNA
- 3 µl 5x iScript reagent
- 11 µl H2O (total volume = 15 µl)
Also set up one no-RT reaction, same as above but using the iScript no-RT control reagent instead.
Run RT reaction as per manufacturer's protocol:
- 25°C 5 min
- 46°C 20 min
- 95°C 1 min
Before proceeding to PCR, add 35 µl H2O to each reaction.
PCR with Taq polymerase and primers flanking the insertion/deletion/mutation
Mix 4 µl virus cDNA with 46 µl of PCR mix containing :
- 0.4 µl dNTP (25 mM)
- 5 µl 10x Standard Taq buffer
- 0.25 µl Taq (5 U/ µl)
- 2 µl Forward primer (10 µM)
- 2 µl Reverse primer (10 µM)
- 36.35 µl dH2O
Note: other RT and polymerase reagents should work just as well. A proofreading polymerase may be preferable if checking for point mutations
Analysis of fragments
Run fragments on agarose gel to check that you have obtained the expected sizes. If appropriate, gel purify the product and send for Sanger sequencing.
