Hi, I am trying to detect Eukaryotic RNA contamination in my bacterial RNA samples. I plan to sequence the bacterial RNA to get a transcriptomic profile. I am aware that qPCR can show if there is any Eukaryotic contamination. But I have not done qPCR or rt-qPCR before and not clear of the theory. Can anyone direct me to a good reference or a protocol that explain how qPCR can help Eukaryotic RNA contamination? Thanks
Hi Nirmani,
there are a lot of genes that you can target to detect human RNA contamination in bacterial samples, but since you planed to run RNAseq on your samples and there will be no way to isolate the contaminant, you should only run RNAseq and during alignment use the human genome and the bacterial genome to get an idea on the amount of waste.
fred
It's not a specific feature of qPCR to detect contaminating eukaryotic RNA. You use primers specific for an eukaryotic RNA sequence, and if you get amplified product, you obviousely detected the presence of eukaryotic RNA. One example of a specific RNA is 18S and 28S rRNA, which is not present in prokaryots but plenty in eukaryots. You can use a qPCR assay for detection, but a standard PCR is as good for that purpose. May be, qPCR can be optimized to give a quicker and easier way to detect a contamination (e.g. when using sequence-specific detection, the observation of an exponential increase of the amplification curve is sufficient to claim a "positive" result, no need for post-PCR processing). But that comes at the cost of some more optimization and higher running costs per PCR (probes are relatively expensive).
qPCR would allow you not only to detect but also to quantify the amount of contamination. The principle is not different to any other qPCR assay. So if this is your aim you should learn this method carefully. There are plenty of books and free online resources where you can start. Feel free to come then back to RG with more specific questions.
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