Hi All,
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?