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Random hexamer vs oligo dT vs gene specific primer for RT


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Poll: Random hexamer vs oligo dT vs gene specific primer for RT (6 member(s) have cast votes)

Please vote

  1. Random primers (2 votes [28.57%])

    Percentage of vote: 28.57%

  2. Oligo dT (5 votes [71.43%])

    Percentage of vote: 71.43%

  3. Gene specific primer (0 votes [0.00%])

    Percentage of vote: 0.00%

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#1 bioforum

bioforum

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Excellent

Posted 13 March 2009 - 08:12 PM

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.

-rajcell-



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.

-indoubt-




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).

-dobbiewalton-



:P 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

-bo_wcl-


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

-John Buckels-


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.

-pBluescript-


#2 netron

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Posted 13 March 2009 - 08:21 PM

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.

#3 Sumpf

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Posted 17 March 2009 - 03:54 AM

First I used only random hexamer but now I am mixing them 1:1 and i got better results.




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