I construct fusion gene stable cell (U2OS and MG63) lines by using lentivirus. But there is an absurd result. I can detec expression of protein (WB), but I cannot detect expression of mRNA (QRT-PCR). Why? Can anyone tell me?
Are you using oligo dT for your reverse transcription?? Lentivirally-encoded transcripts are not polyadenylated. You need to use random hexamers or transcript-specific primers for the RT step.
Thanks for your answer.
I used PrimeScript™ RT reagent Kit with gDNA Eraser(TaKaRa) ,including oligo dT and random hexamers.
I did an immunofluorescent expriment today . I could detect fluorescence in fusion cell lines , but vector cell lines not. It's a rational result.
mRNA level of this fusion gene may be too low to detect in U2OS and MG63.
Should I exchange a carrier to construct the stable cell lines ?
Li- I don't follow your last question; what do you mean by "exchange a carrier?" If a significant fraction of cells infected with your lentivirus are positive for your gene of interest by immunofluorescence, I'm not sure why you want to quantify the mRNA. Protein-based metrics are usually more relevant for demonstrating trans-gene expression.
Maybe the oligos used for your qPCR are no good, or you need to add more cDNA to your reactions; both are trivial reasons for why mRNA levels would be very low. You should check that your primers work by adding a small amount of lentiviral plasmid as a positive control condition in your qPCR.
I'm sorry for typing a wrong word . I mean change a vector to construct another stable cell line.
I want to quantify the mRNA level because I think QPCR is a more accurate method to demonstrating gene expression.
Do you mean that I have demonstrated my stable cell lines without result of mRNA level
Yes, I would say that you don't need the qPCR data. If this gene is not expressed endogenously, immunofluorescence and Western blot are more informative than qPCR. mRNA and protein levels are not always linearly correlated, and therefore the quantification of mRNA by qPCR only tells you that you have a strong promoter for transcription, but not that you are getting robust protein expression.
I don't think that changing to a different viral vector will resolve this problem. Try ordering new oligos. Make sure the Tm >55C, and the amplicon is around 75-150bp. Also, as mentioned above, have a positive control with plasmid DNA that contains the cDNA of interest in your qPCR (you can even run a standard curve using a dilution series of this template).
Hi, Zhong, I have the same question as you. I made the stably cell lines using lenti-virus (pCDH as vector) overexpressing the gene I am interested in. however, the mRNA level of the gene is 20-fold lower than empty vector control detected by qPCR, while the protein level is perfect by Western blotting. So I am really confused now, did you fix the problem? thank you very much.
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