I'm trying to increase sensitivity (detection) for a gene expression assay. The more RNA or cDNA I add the more detection I get. I need to be able to detect at really low parasitemia, so the thought is that if I keep adding more material, then I might eventually be able to get some detection. My master mix gives me room for about 10ul of material+water to add per reaction. Would there be a problem if I decide to add all 10ul of my material? How would I be able to detect if there is a problem?
PCR Inhibitors can be transferred from tissues during extraction or from kits. If your sample is not diluted enough, you may get more inhibitors than the reaction can handle. If you do hit a limit where your PCR does poorly, that might be a reason. There are many PCR troubleshooting guides online that talk about PCR inhibitors.
I would also refer to your Taq product guide to what the maximum ng of template is recommended for your reaction, then you can calculate that from your RNA concentration.
Thanks for replying Michael. My case is a little complicated. I'm interested in parasites that have been taken up by mosquitoes during their feed. I extract RNA from the whole mosquito, and then nanodrop. Unfortunately the nanodrop gives me a concentration, but I don't know how much of that is actually from the parasite. Most of it I know should belong to the mosquito. So, for example, if I calculate to add 150ng of RNA, I know I'm not actually adding 150ng of my parasite. I wanted to see how much RNA I can keep on adding without running into problems, but I wasn't sure if I would be able to detect what these problems might be.
I agree with Michal. Usually the kit indicates what is the maximum amount from the cDNA that you can load into your PCR reaction tubes so that it doesn't inhibit the PCR reaction. Like for example in my case I use iScript cDNA synthesis kit, they indicate that "the maximum amount of cDNA reaction that is recommended for downstream PCR is one-tenth of the reaction volume".
O. Marte, I have not specifically worked with parasitic RNA, but I have a few ideas that might help.
1. Do you have a positive control? In other words, is there a target transcript that you could detect by qPCR that would at least tell you that your RNA prep contains parasite RNA?
2. You may look into designing a custom oligo probe for the capture/enrichment of your target of interest. The probe could be conjugated with a moiety (avidin, GST, magnet) for pulldown/purification. I would design a probe for a positive control as well and perform the pulldown for both transcripts in the same tube.
3. Somewhat similar to #2 above, you can use gene specific oligos to make cDNA only to your gene(s) of interest, thus effectively enriching for your transcript targets. The other transcripts get digested by the RNAse step in the cDNA prep protocol.
4. Any chance that an antibody exists to detect the protein that is encoded by your transcript of interest? I doubt many antibodies exist for parasite proteins, but worth considering anyway.
If you just want to know if the transcript is present, I think it would be sufficient to run a PCR with the enriched RNA from #2 above. Alternatively, you could run a qPCR with it.
By the way, I typically avoid using large amount of RNA/cDNA in my qPCR assay as you may negatively impact your reaction efficiencies, which in turn negatively impacts your ability to detect your transcript. So you may actually be reducing the chance that you will detect the transcript by using large amounts of RNA/cDNA in your reactions. I prepare cDNA from 100ng of total RNA using poly-dT oligos and then only use about 0.25uL (or about 1.25ng of RNA input) per 10uL qPCR reaction.
Best.
I co-authored the attached paper which gives you some good parameters for your situation. In this case, the parasite in the mosquito was Brugia malayi.
The key thing for the qRT-PCR was to do a dilution study of a mixture of a portion of all experimental samples; starting at full-strength and going out until no signal was found. (This preliminary study was performed on a parasite reference gene). From there, the standard curve ranges were established, and a good qRT-PCR pre-dilution for all samples was identified - a dilution that fell within each target standard curve range.
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