Hello
I am working on a gene to detect in various cell lines.
The gene was detected by Western blot and siRNA was also worked fine (The gene was silenced by specific siRNA transfection), but not detected in RT-PCR.
I tried to design different primers and did RT-PCR but not detected either.
The gene has high GC content ~70%. Do you think this is problem for PCR?
Does anyone have good suggestion for this issue? or Have you ever heard a gene undetectable in RT-PCR but detectable in Western blot?
Thanks.
Dear Sang, firstly you must rule out all the technical issues that are potential reasons to bash any qPCR reaction without giving us any logic often that what actually went wrong. Many of those are already well explained by my respected colleagues just above, which range from your RNA extraction phase till the final qPCR step.
But here, I would like to focus at your question in an another dimension. Usually at low or even no mRNA detectable signals, we may have good strength for its respective protein signal, as protein has the tendency to store, so at westerns we always or mostly get their signals, while, mRNA level in a cell is subject to its turnover, degradation, possible inhibitory actions of cytoplasmic miRNAs over your desired mRNA translation (which is one of the hottest topics of this era), partially blockage of mRNA by any external stimuli or stress, or its extreme low expression in the case of low expressing genes. So, one has to take in account these tricky exhaustive issues in mind. But just remember that the final language of genome is the protein, which of course far more stable than mRNA. Hope it clicks.
You mean, your primer has high GC content?
If the problem is not on primers, the most important steps is during extration. Try to now rna concentration or dna after RT by spectra uv
Regards
Such observations might happen with certain plausible causes.
1. if the antibodies are designed against the entire protein and one or both of the primers are designed on an alternatively spliced exon that is absent from the mature mRNA.
2. You could also review the cDNA preparation method. Id oligo dT used or random hexamers/nonamers. certain transcripts may lack poly A tail and may not be represented in the cDNA population although present in RNA pool. A Northern hybridization may help probe both the reasons.
meanwhile, amplification problems due to high GC can be verified as well as optimized by using genomic DNA or cloned plasmid DNA as template.
Yes high GC content in target might hinder PCR efficiency
that being so
- start PCR with single denaturing cycle of 96C for 5 min
- If using standard Taq denature per cycle at 96c for 40 sec
- also add 10% DMSO to your PCR reaction
- If using Taq add 1ul per reaction instead of usual 0.
- Better still use a highly processive polymerase like phusion
- If using phusion denature at 98C for 10 sec per cycle
- if your primers have a Tm such that your current annealing temp is about 55C to 60C perform std 3 step PCR with an extension cycle of 72C after separate annealing cycle
- better still design GC rich primers such that your Tm is about 65-70C and then perform 2 step PCR in which annealing and extension are performed in the same cycle at 65C; that is a PCR cycle consisting of 96C for 40sec with standard taq (1ul) or 98C for 10sec if phusion followed by 1 cycle of 65C for upto 2 min (per kb of product: that is 65C for 2 min for 1kb or 65C for 1 min for products at or below 500bp
- add 5 extra cycles to your PCR: that is for example 35 cycles instead of 30
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