Does anyone have experience doing RT-qPCR on RNA from nuclei, rather than whole cells? Does this work well using standard protocols/primers/probes, or are modifications needed? More details below:
I am hoping to do RT-qPCR on 3 nuclei populations I isolated with FANS to validate whether they are indeed from the cell types I was interested in (neurons, oligodendrocytes, microglia from human brain tissue). My lab uses one of the TaqMan qPCR master mixes and thus, we usually just order the pre-made primer/probe sets that they offer. However, I'm concerned that maybe these primers/probes won't work as well on cDNA from nuclei as opposed to whole cells, since there is a lower proportion of mature mRNAs in the nucleus and more pre-mRNAs, and these primers/probes tend to be specific to the reverse-transcribed, fully spliced mRNAs, overlapping exon junctions to avoid gDNA amplification. I'm not sure if this difference in RNA between the nucleus and cytoplasm is really big enough that it would produce weak results.
I was hoping to target these genes for each population: ENO2 or MAP2 - neurons, OLIG2 or SOX10 - oligodendrocytes, AIF1 - microglia. I imagine there are a few different ways I can approach this experiment, but the simplest course of action would be to just make sure the genes are highly expressed enough in each cell type that it wouldn't really matter is there was less mature mRNA in the nucleus.
In your experience or to your knowledge, should it be okay to just order the premade primers/probes and use those on nuclei? Are the marker genes I listed above highly expressed enough that I should be fine? Thanks in advance!
(Notes: I'm not concerned about testing whether what I have are truly nuclei vs. whole cells, as this is not relevant to my research question, so I'm not looking for universal "nuclear" markers. All I care about is which cell types the nuclei came from.)
Can it work? Yes.
Will it work? Maybe.
Nuclei are actually host to a surprising number of mature transcripts: using oligodT priming alone for RT will generally still allow you to robustly detect many genes. Single nuclear RNAseq approaches rely on exactly this.
But, not all genes are the same: some transcripts are expressed at lower levels, and exported more rapidly, than others. So your mileage may vary, and your expression levels within nuclei might not match those in bulk.
I would not suggest attempting to detect unspliced transcripts, especially not initially: introns are often removed pretty promptly (even co-transcriptionally) and genomic DNA contamination is a real issue when working with nuclei. Sticking with known Taqman probes you have already validated in other scenarios is a smarter move.
Also, bear in mind that you might also need a lot of nuclei to robustly detect mRNAs via qPCR.
If you're lysing and extracting RNA from them first, probably favour column methods over cheap/cheerful trizol+precipitation.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts