Hey,
I am doing RT PCR with both TAQ and SYBER primers.
My normalizing genes are great, unify, and perfect.
However OTC, even in HepG2 cell line, is not unify and raises in the same technical replication in a variability of even 1/5-2 cycles (while normalizing gene is unify w/o any problems).
happens in Taq and syber.
I tried to raise DNA concentration, and also various HepG2 lysis from different passages.
Any idea how to solve this?
- Are your primers pan specific in terms of spliceforms ?
- If not design primers which target exon exon boundaries to avoid spurious genomic DNA reactions and also specific to an exon junction common to all isoforms to standardise amplification
- http://journals.plos.org/plosone/article/figures?id=10.1371/journal.pone.0122966
High Ct variation in same technical replicate is usually seen when the Ct value is high and the copy no. is very low or gene expression very low.
Optimize the PCR condition like primer conc., Ta. For optimization of primer concentration run one RT PCR with different concentration combination of forward and reverse.
See the reference and attach file
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706253/
Good luck
thanks for your answer! Are you familiar with a file such as the above, for TAQ primers?
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