I want to know the artificial gene or strain which can be use as a DNA/RNA extraction control, and will lead you to RT PCR testing for any viral DNA or RNA.
Hi,
Kindly follow links below for some suggestions
about RNA/DNA extraction I thought about the RNA and DNA spike, (an example http://www.bioline.com/sg/dna-extraction-control-670.html, http://www.bioline.com/au/rna-extraction-control-670.html), then you can buy the primers to check the spike extracted throughout the RT and PCR. I used this method for microRNAs analysis in serum (example http://www.exiqon.com/rna-spike-in-kit)
You have to study kits and strategies for the different samples and choose the companies that offer you the best at "low cost". Hope this can help you,
https://www.bioline.com/downloads/dl/file/id/3320/dna_rna_extraction_controls-factsheet.pdf
good luck
this link is useful
https://www.thermofisher.com/iq/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/basic-principles-rt-qpcr.html
regrards
Hi,
Kindly follow links below for some suggestions
about RNA/DNA extraction I thought about the RNA and DNA spike, (an example http://www.bioline.com/sg/dna-extraction-control-670.html, http://www.bioline.com/au/rna-extraction-control-670.html), then you can buy the primers to check the spike extracted throughout the RT and PCR. I used this method for microRNAs analysis in serum (example http://www.exiqon.com/rna-spike-in-kit)
You have to study kits and strategies for the different samples and choose the companies that offer you the best at "low cost". Hope this can help you,
https://www.bioline.com/downloads/dl/file/id/3320/dna_rna_extraction_controls-factsheet.pdf
good luck
Its reasonable that you make an IPC (internal process control) that can be spiked into your crude extract which is well characterised. For RNA we use phage MS2 that is grown in E.coli hosts. You can calculate the eaxact number of viral copies by the TOPagar method and store your bulk at 4 oC (virus keep their infectivity for more than a year) and at -80 oC (for reproducible results). Then process the virus throught your purification system and calculate the recovery by RT-qPCR with MS2 specific primers/probe. Charactise your MS2 batch by a 10 fold dilution series. Then you know the relation Cq and viral count. It is impotant to do this in two steps. FIRST dilute MS2 in water, SECOND spike MS2 in negative matrix, perform the same diluiton serie, and then follow your extraction protocol and RT-qPCR. This allows you detect the influence of your matrix on extraction. Next to this you can compare various purification methods up to exact genome copy numbers. The only assumption is that isolation of the viral MS2 genomic RNA can be used as a model for your target at interest.
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