Hi guys,
So I am working with two colorectal cell lines (HCT116 and HT29). I extracted RNA, converted to cDNA and ran PCR for my gene of interest. Then also extracted protein and did a WB for the gene of interest.
Compared to HCT116, for the PCR, the gene signal was stronger in HT29 but for western blot, the bands were fainter in HT29. Can anybody explain what could be going on?
Given you simply stated "you ran a PCR" and referenced band intensity, I would assume you are not assessing transcript levels by any truly quantitative means.
The intensity of PCR bands on a gel is notoriously difficult to interpret quantitatively: you need to be certain that you're capturing the amplicon before it reaches saturation but after it becomes detectable via gel, and be similarly certain there are no inhibitors present (or substrates absent) that might impair the later stages of the amplification.
And then you need to do the same for some reliable internal standard (you do not mention this).
I would recommend you perform a qPCR (or other truly quantitative metric) for your cDNA before you make any assumptions as to expression level, and make sure you normalize your data to (ideally) two or more solid reference genes.
Overall, there is no empirical reason why this effect shouldn't be real anyway: RNA levels need not correlate with protein levels, even for the same gene in different cell lines. microRNAs can hinder translation, such that even high levels of transcript result in little or no protein product, while highly stable, actively translated mRNAs can generate large quantities of protein from relatively few transcripts.
- I agree with Jon: different cell lines might process the protein in different ways so steady state protein levels detected in a snap shot assay like western blotting might not correlate with relative band intensities by RT PCR
- Does the HT29 show stronger smaller bands common to both lines by western blotting: if so - as opposed to just a single band of the predicted size - then that might imply you are seeing augmented in vivo processing into smaller degradation products with HT29
- It really comes down to rate of synthesis versus rate of removal; that is steady state levels of the protein
- in addition RNA transcript processing might vary during time if cyle so depending when you assayed your mRNA levels by RT PCR this couls also alter things
Their are chances of RNA transcript forming but no protein formation from that trancript.
Gene expression (i.e., mRNA/transcript level) using PCR is not final product of the central dogma. Genes are highly regulated by several factors, so their up-regulation and down-regulation is much more context-dependent.
Now, after RNA, its not directly the proteins which forms, RNA undergoes various post-transcriptional modifications, which will definitely regulates the corresponding protein formation. So, Gene expression and next protein expression can't be same. Even, epigenetics (modification/changes) is matter of consideration.
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