Using pentadecamer primers for reverse transcription - Better than hexamers, oligo d(T)? (Aug/02/2006 )

Has anyone used random pentadecamer primers for reverse transcription? Stangegaard et al. describes in Biotechniques May 2006 that, "Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA". I wanted to know if anyone else has had success (i.e. over random hexamers). Also, where can you order random pentadecamer primers?

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Okay so I found one company, Mobix Labs, offers random pentadecamer primers
http://www.science.mcmaster.ca/mobixlab/ol...primerform.html

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We know that the ordering of the random pentadecamers was not so well described in the paper. The reason being it is just so simple. However we did get quite many mails regarding this issue. Therefore we wrote an addendum to the paper addressing this issue.

The addendum was published in the July issue of Biotechniques (BioTechniques Vol. 41, No. 1: pp 50).

In short: You order random 12 or 15 primers from your conventional oligo vendor by ordering a wobble oligo (usually denoted N) of the desired length (i.e. NNN NNN will give you random hexamers while NNN NNN NNN NNN NNN will give you random pentadecamers)

From Figure 1 of the original paper (Biotechniques. 2006 May;40(5):649-57) you can see that ordering say random hexamers or nonamers this way results in yields comparable to the Invitrogen random primer formulation. Besides, the Invitrogen random primer formulation is quite expensive, while ordering short oligos this way usually is significantly cheaper. You can find a calculation example in the addendum Biotechniques (BioTechniques Vol. 41, No. 1: pp 50).

Good luck with you RTs
Michael Stangegaard

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Have you tried mixing in random pentadecamers and oligo d(T) primers? I'm about to make some cDNA and want to be as comprehensive as possible.

Cheers,

CambMatt

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No we have not tried that. I did try out oligo d(T)s with the form T10-NN and T20-NN (a stretch of 10 or 20 T with two wobble in the end). The idea was to increase the binding of the poly T oligo and to direct it to the 3' end. However the results was not very good.

I would spend my time on the random priming. Perhaps mixing nonamers with R12 and R15 - maybe also R18. If you order them desaltet (NOT HPLC) purified they are both cheaper and you get the shorter partially synthesized oligos.

But using either R12 or R15 will get you a yield close to 100% according to our calculations if your reaction is optimized ;-D

Good luck

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That's excellent. But is there not a bias away from the 3' end of genes when using random primers as opposed to oligo d(T)?

CambMatt

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Hi,
On the contrary. You will most likely experience 3’ bias when using oligo dT priming. During the RT the polymerase will fall off. As random priming initiates transcription at random positions in the RNA template this will not occur. However using random hexamers rather than R12 or R15 might give you a bias as the primer melts away during the reaction due to the low melting temperature of the short duplex.

Stangegaard

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Hi there!
I would greatly appreciate if any of your help me with a solubility problem using 15-mer random primers.
I recently ordered 15-mer random primers following the indications in the addendum to Stangegaard's Biotechniques paper (1 umol scale and desalted, Sigma-Genosys). When I tried to rehydrate them in DEPC-treated water at a final conc. of 1.34 nmol/ul, I got a turbid white precipitate that I cannot manage to dissolve. Have any of you had this problem before? If so, how did you solve it?
Thank you very much in advance for your time and kind help,

Best wishes,

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