Aug 13, 2025
Version 2
RSV standard and copyback genomes PCR Protocols V.2
- Carolina Lopez1
- 1Washington University
Protocol Citation: Carolina Lopez 2025. RSV standard and copyback genomes PCR Protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokkndl4o/v2Version created by Carolina Lopez
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 13, 2025
Last Modified: August 13, 2025
Protocol Integer ID: 224645
Keywords: respiratory syncytial virus, copyback genomes by pcr, rsv standard, copyback genome, pcr protocols protocol for the detection, rsv, pcr protocols protocol, pcr, copyback, virus
Abstract
Protocol for the detection of the Respiratory Syncytial Virus (RSV) standard and copyback genomes by PCR
Materials
Reverse Transcription (RT)
PCR
Troubleshooting
Before start
READ ME FIRST!
What is a PCR hood/cabinet?
Our PCR hood is a dead air box that prevents contamination between
samples by minimizing air circulation in the environment. The UV light helps
decontaminate the workspace between uses.
When should you use the PCR hood?
cbVGs are highly stable in their RNA form. In addition, some species in highly abundant and once they are reverse transcribed into cDNA, you have a lot of very stable cDNA in the environment. Therefore, to avoid contaminations in PCRs and sequencing cbVG cDNA should be handled in the PCR hood to minimize contamination. Specifically,
After performing cbVG-specific RT reactions, RNaseH should be added in the PCR hood after RT. The aerosol that occurs when opening the cDNA-containing PCR tubes is a major source of contamination.
After PCR master mix is aliquoted at the bench, cDNA should be added to each reaction in the PCR hood.
Note, PCR hoods are a dedicated workspace designed to minimize DNA contamination; they are not RNase-free environments, so they are not the best place to handle RNA. RNases are ubiquitous in the environment and even miniscule amounts can quickly degrade an RNA sample. PCR amplifies huge numbers of the target DNA, and these amplicons can easily contaminate subsequent reactions if proper spatial separation and clean practices are not maintained.
Tips for using the PCR hood:
The PCR hood is not a Biosafety Cabinet. PCR cabinets do not protect the operator; they only protect samples inside the work zone.
Do not store unnecessary things in the PCR hood, as this can reduce containment.
The workstation should be wiped down with disinfectant prior to and after use, and all external items should be disinfected before they enter the PCR hood (things like tube racks).
Pipettes should be disinfected before and after use: spray disinfectant (10% bleach) on a paper towel and wipe them down (do not spray the pipettes directly).
When applicable, minimize contamination risk by processing LD samples first, followed by HD or higher concentration samples.
Do not reach over your samples. This can contaminate your product.
When finished, turn on the UV light to decontaminate the workspace between uses and eliminate any airborne contaminants (for our hood, the knob is broken so you need to use the tool that is usually sitting on top to turn it on).
Change gloves immediately when finished to reduce contamination to other areas in the lab.
RSV standard and copyback genomes PCR Protocols
*The “Big” DVG design can detect cbVGs with break before 14882 and rejoin before 15112 (RSVA2). The “Small” DVG design can detect cbVGs with break before 15112 and rejoin before 15194 (RSVA2). The “Small” DVG design should theoretically also pick up big DVGs, but bigger amplicons do not get amplified as well. “gRSV rev” has been tested as an RT primer for both “Big” and “Small” DVG design, however, gave an unspecific amplicon with the “Big” DVG design at around 500bp. As a consequence, two separate RT and PCR reactions must be run when wanting to look for both big DVGs and small DVGs. For most experiments, it is enough to only use the “Big” DVG design. RSV Genome can also be detected using this protocol when using the Genome primers. Primer locations are 387-410 and 894-918 (RSVA2), however not an optimal match (seems to have been designed for RSV B?).
Procedure
Reverse Transcription (RT): *RT reaction can be completed in a 10 μL reaction or a 20 μL reaction. The 10 μL reaction is preferred, if RNA concentrations allow, to help conserve reagents.
1. Start with 500 ng of RNA diluted in dH2O (range 200-2000ng (background increases with more RNA))
10 µL reaction: dilute in 4 µL dH2O
20 µL reaction: dilute in 8 µL dH2O
2. Add primer (2 micromolar (µM) )
10 µL reaction: 0.5 µL
20 µL reaction: 1 µL
3. Add dNTPs
10 µL reaction: 0.5 µL
20 µL reaction: 1 µL
4. Incubate at 65 °C for 00:10:00
Program in PCR Machine: RT-DI1
5. Prepare the mix
6. Add mix to sample
10 µL reaction: 5 µL
20 µL reaction: 10 µL
7. Incubate at 50 °C for 00:50:00 then 85 °C for 00:05:00
Program in PCR Machine: RT-DI2
8. After program is finished keep at -20 °C for at least 00:20:00
9. Spin down tubes in mini microcentrifuge
10. Add RNase H (add in the PCR hood! DVGs are very stable!)
10 µL reaction: 0.5 µL
20 µL reaction: 1 µL
11. Incubate at 37 °C for 00:20:00
Program in PCR Machine: RNaseH
12. Keep in -20 °C for at least 00:20:00 before moving on to the PCR
2h 5m
PCR
1. Prepare master mix (can be done on bench)
*Volumes below are for when 1 μL of cDNA is used. Adjust dH2O volume accordingly if more cDNA is added. Up to 4 μL cDNA can be added but increases background. Total reaction volume should equal 25 μL.
2. Add 24 µL of master mix to PCR tubes (can be done on bench)
3. Thaw cDNA samples and spin down in mini microcentrifuge
4. Add 1 µL of cDNA sample to master mix (add in PCR hood! DVGs are very stable!)
5. Run PCR
Program in PCR Macine: RSVDI233
Gel Electrophoresis:
30m
1. Prepare 1% agarose gel
1 g pure agarose in 100 mL of 1X TAE buffer
Microwave until agarose solution dissolves completely (00:02:00 )
Let agarose solution cool before adding Ethidium bromide (if you can keep your fingers on the flask without burning, then it is at an appropriate temperature)
Add 1-5 µL of Ethidium bromide and mix by swirling flask
2. Pour agarose solution into gel cast (remember to put in the well comb)
3. Let the agarose solidify (wait at least 00:30:00 )
4. Place gel in electrophoresis chamber containing 1X TAE buffer (make sure buffer covers the gel)
5. Load ladder and samples to wells
6 µL of Ladder
Ladder stock recipe:
100 µL GeneRuler 100 bp Plus DNA ladder from Thermo Scientific: SM0321
100 µL DNA Gel Loading Dye (6X) from Thermo Scientific: R0611
400 µL dH2O
30 µL of Sample + Loading dye mix (dilutes from 6X to 1X)
5 µL of DNA Gel Loading Dye (6X) from Thermo Scientific: R0611
25 µL of sample
6. Run at 110 volts for 00:30:00
7. Analyze gel bands
Band sizes and sequences were confirmed with Sanger sequencing.
1h 2m