Hello,
I want to check the expression levels of 4 different genes in a bacterial cell.I have isolated total RNA and intend to synthesis cDNA for transcriptional analysis.I am confused whether to use random primers or sequence specific primers for this. Will adding specific primers for all the 4 genes at a time be efficient? I have a limited amount of reagents.
Thank you.
You've extracted total RNA, so why not simply use random priming to effectively make 'total cDNA'?
With gene specific primers, you'll just make cDNA of your transcripts of interest.
With random primers, you'll make cDNA of your transcripts of interest and all the other RNA.
All you'll do by using gene-specific primers is render your cDNA of limited utility. Assuming your transcripts of interest are not expressed at vanishingly low levels, there is no real advantage to be gained by deliberately excluding the vast majority of your RNA from the cDNA synthesis reaction.
With no doubt, go for random primers to synthesize cDNA. All the best!
Thank you, for the answers.
I just have to study the expression levels of genes of my interest. So why should I make cDNA of all other RNA in which I am not interested? Moreover, random primers results in short length cDNA fragments. Instead, I could use sequence specific primers for the four specific genes which would yield cDNA in which I am interested. What's the difference between the two?
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