expression of npcRNA - (Aug/08/2006 )
I want to see the expression of one non protein coding RNA (npcRNA) in a cell line(HeLa). It is actually derived from an intronic sequence of a human protein. I did PCR amplification from previous exon, intron and next exon. I cloned in pcDNA3.1+(CMV promotor) between BamHI and XhoI. Transfected in HeLa, but I am not seeing the npcRNA product after northern. Can you suugest some thing for this.
Thanks in advance
If you can find the npcRNA from the control cell line (before cloning) but not after you're 'losing' something after cloning. This could be the result of a lot of factors.
Are the flanking exons related or included in the same endogenous transcript? If so include the entire genomic sequence of that gene in the cloning fragment. It may require the additional sequence for post transcriptional processing that was missed in your first attempt.
But you could have other technical issues such as poor pcr amplification (try sequencing your fragment). Poor expression (verify that the CMV promoter is intact and working after transfection).