Random hexamer vs oligo dT vs gene specific primer for RT - which do you use most? (old and useful thread) (Mar/13/2009 )
Dear fellow researchers,
Could you tell us what type of primer (random hexamer vs oligo dT vs gene specific primer) you use most for RT reaction and why.
you use gene specific primers when you use one-step Rt-qPCR. you use random hexamer or oligo dT or a combination of them when you use two-step Rt-qPCR. one-step Rt-qPCR can be used when there is one target you want to amplify while two-step Rt-qPCR can be used when you want to amplify more than one target. you save alot of samples using two-step Rt-qPCR because the same Rt sample can be used many times.
Oligos DT- mRNA specific. Reduces your cDNA complexity and amount since poly A+ RNA makes 1%-2% of total RNA (vs. random hexamer). Recommended when performing RT-PCR of a new mRNA target. Oligos DT produced RT-PCR products more consistently than random hex or GSPs (GSPs sometime fail to prime cDNA synthesis even when functional as PCR primers).
Dear all professionals,
Thanks for all the nice answer. I learned a lot from it. How about the combination of random primer and oligo dt primer? When do we need to use it?
A fresh Mphil student
I sometimes do RT-PCR on the ribosomal spacers or 5S rRNA, in which case I use random pimers. Other times I need to look at full length transcripts or long transcripts, then I use oligo dT. Other times I need to do one step RTPCR or look at very rare transcripts, then I use GSP
Oh what a varied scientific life I lead
For real time pcr, I always use random hexamers.
Full length transcripts are not needed and you get much more converstion of RNA to cDNA thus you carrry more mass over. Plus, it works well. I cannot say the same for oligiodT.
I only use oligo(dT) primers for all my RT reaction unless I am converting RNA known not to have poly(A) tails. I believe oligo dT primers is more specific to mRNA.
First I used only random hexamer but now I am mixing them 1:1 and i got better results.