Is it necessary to isolate nuclei from cells and use this RNA for qPCR? Or can specific primers be designed in intronic regions so that only unprocessed RNA will be picked, and use whole cell RNA as the starting material?
If the former, are can anyone suggest efficient nuclei isolation methods for B cells?
Hi,
Primers which span intronic and exonic regions can measure pre-mRNA but also measure DNA.
Maybe use DNase?
Asif
Hello Devi
that's an interesting proposition. The only thing I will say that is you design primers to pure intronic regions you will of course detect genomic DNA as well as unprocessed primary RNA; whether using purified nuclei or whole cell extracts
What I would say is that if you digest your genomic DNA with DNAse I then you will not detect genomic DNA. I don't know if you require just a nuclear enriched fraction for this procedure to work efficiently
What I would say with ref to specificity is that you will need to digest your gDNA derived from the most appropriate fraction. Recombinant DNAse I is ideal for this as you can digest your RNA @ 37C for upto 1 hour and not incur RNA degradation which can be an issue with purified DNAse I fractions. Personally, having worked with various types of DNAse I the rDNase I known as Turbo, made by PE and sourced from either PE or Fisher scientific works extremely well in my experience:
https://www.thermofisher.com/order/catalog/product/AM2238
Thanks for the suggestions. Whether a whole cell extract or a nuclear prep, DNA will interfere and I shall definitely do a DNase treatment before cDNA synthesis. Literature suggests a nuclear prep before the RNA quantitation. I was asking if I could take a short cut around the isolation.
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