Random hexamer vs oligo dT vs gene specific primer for RT - which do you use most? (Jun/05/2005 )

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.

Thanks.

Raj.

---

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
Victor

---

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.

---