I want to detect early expression of IL2 by qRT-PCR. I was not successful using random hexameric primers at the RT step and SYBR for detection. what can I do to improve my sensitivity?
What difference does it make if I take oligo dT for RT or a mixture of Oligo dT and random primers?
Oligo dT will amplify all mRNA molecules that have a polyA tail. Random primers will amplify a subset of mRNAs and may not be efficient at amplifying your specific target. Try just oligo dT first and see if you get better results.
Oligo-dT will bias your reverse transcription towards the polyA-tail end of mRNA, ie not a good idea if your target is a very long RNA (no idea if IL2 is long or short). If you designed your PCR primers to detect >2-3 kb away from polyA-tail then you've introduced a bias in your RT that will underestimate your expression, particularly if your reference genes have primers close to polyA-tail.
Random hexamers will prime RT at their random target sites which should be random within the whole of your mRNA, so definitly preferred for longer targets.
Mixture of both is meant to be robust and cover both ways of amplification...
You may have very low expression of target, target-specific primers in the RT step are meant to improve specificity... but then you can't use reference genes on the same sample...
You can always introduce a preamplification step as reviewed in this paper:
https://www.ncbi.nlm.nih.gov/pubmed/26132215
Warning about random hexamers: These are often used to amplify non-polyA transcripts, so your target may end up being underrepresented in the cDNA population compared to if you use oligo-dT. I've used random hexamers in the past to amplify miRNA transcripts.
Our lab uses the Roche UPL system to study epigenetically repressed genes. It is based on a hydrolysis probe, but much less expensive than Taqman and similar DNA-based probes. In UPL, a short locked nucleic acid (LNA) dye-labeled probe binds to your target, and a pair of primers surrounding the probe hydrolyze the probe, freeing the dye from the quencher. We have used these successfully for genes that have a basal silenced state (Polycomb-silenced) and become up-regulated after epigenetic modification (Nyer et al. 2017 Nature Genomic Medicine).
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