5 replies to this topic

[kblee]

Hi all,

Recently I transient transfected a plasmid which carry my target gene (size 7kb) in to mammalian cells (LO2) but I can't detect any protein changes through western blot.
So I try to use RTPCR to detect the mRNA level of my target gene if it rise up after transient.
But one of my labmates ask me how can I make sure the RTPCR has fold changes due to my target gene from mRNA but not the plasmid template in the cells.
Simply  how can I make sure the RTPCR is amplifying mRNA but not DNA? (the target gene in the plasmid is translated region from cDNA)
Thanks in advance!

kb

[prabhubct]

Did your target gene has introns too? if yes you are going to get more lenght of amplicon with DNA than one with mRNA. You may see difference in melting curves.

Edited by prabhubct, 13 September 2012 - 09:10 AM.

[pcrman]

Yes, you can use RT-PCR to verify the expression of your exogenously expressed genes, but you have to treat your RNA thoroughly with DNase to make sure no amplification is from DNA. If your transfection worked, you may obtain hundreds of fold increase in expression.

[prabhubct]

''the target gene in the plasmid is translated region from cDNA''  It's only exon region of gene.  yes, you do need to treat RNA with DNase too.

[kblee]

Thanks for the prompt reply   Can I collect the cells doing western blot and RTPCR after 72hr of transfection? Or I have to collect the cell for RTPCR after 24hr? 48hr?

[pcrman]

24 hr may be too short, although at that time, the vector is already expressed. I would say 48-72 hrs will be better.