I want to PCR a 2kb long segment of a gene out of the cDNA made from total RNA extract but have been experiencing many weird problems so I hope the brilliant minds here can shed some light on it.
The total RNA was extracted from ~100 whole embryos (~1000 cells each) of an invertebrate marine species using QIAGEN RNeasy Plus mini kit eluted with 40ul RNase free water. Nanodrop gives a low reading and a weak absorption peak (~10ng/ul). Gel analysis of 3ul such RNA yielded no bands (not even the ribosomal RNAs). The RNA sample have been heated at 65C for 5min before electrophoresis to eliminate hairpin structures.
The cDNA was obtained using iScript reverse-transcriptase, primed by random hexamers. Curiously, the same cDNA gives weird results when taken as template to amplify specific gene products:
for some proteases, very bright bands.
for some transcription factors highly expressed at the same time, no bands at all.
for a housekeeping gene in the species (ubiquitin), no bands.
the regions of these gene i am amplifying are all 2kb, cycler conditions and PCR rxn mix followed factory recommended protocols. In all cases the primers, enzymes (Advantage 2 from Clontech) and other reagents were tested and they work fine with controls.
My question: to me everything points to the quality of the cDNA, maybe low concentration of the gene products present in the total sample. But why some genes can be amplified but others not?
Disclaimer: I normally work with plasmid/genomic DNA, so might be wrong in my suggestions.
Possible problems may be:
1. low amount of mRNA. You don’t have that many cells total and you can’t be sure that mRNA is stable for your genes of interest. Possible solution — increase number of cycles.
2. Primers. If you used primers many times before, freezing-thawing can affect their quality. Making new primers from stocks can help. I assume you checked specificity but might be worth looking again.
3. GC content. If primers/genes of interest have high GC content or plenty of hairpins, specific conditions should be applied. In case of normal PCR it means special buffers, DMSO/BSA, etc.
4. Chealating agents. Don’t know if any of your solutions have them. If ‘yes’, Mg2+ concentration should be adjusted.