I extracted RNA from human saliva samples but the amount of bacterial (basically not human) RNA is very high: despite the high RNA concentration (Nanodrop quantification), after real-time PCR the ct values of the housekeeping genes are very high. I assume human RNA is only a small fraction of the total.
I would like to know if there is a method to separate bacterial from human RNA in my samples. I was thinking about a retrotranscription reaction from RNA to cDNA; could this reaction be human-specific? which primers should I use?
Thank you in advance
1. Bacteria don't have introns. Try to design primer from splicing junction and check in NCBI primer blast whether the designed primer would still bind to bacterial RNA or not.
If the 1st option does not work for you then can try the 2nd option below-
2. Poly(A) tail present in mRNA of Eukaryotes, but not in Bacteria. Design a primer from poly(A) region of mRNA and another one from the coding region of mRNA, thus the set would only amplify mRNA that are from human.
Hi.
If you want to do gene expression , then you can separate your host mRNA from the bacterial RNA by using polyA tail-based techniques because bacterial mRNA is not polyadenilated.
You can also try adding gene-specific primers if your cDNA synthesis kit is compatible with this.
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