TruSeq DNA adapters in RNA-seq prep... concentration?
 

Hi Everyone,

I'm in a situation whereby I need to use two extra adapters than the 6 adapters that are currently shipped with each set of the TruSeq RNA-seq prep kit and at the moment we only have one set. We have both sets of adapters for the TruSeq Genomic DNA prep kit (i.e. all 12 adapters).

I know the Genomic DNA and RNA-seq adapters are the same, but the concentration is different. Has anyone used the adapters from the DNA kit in the RNA sample prep? I think it used to be a 10 fold dilution of adapters using the 'old', non TruSeq kits.

Cheers,

Scott.

Can't you just check using absorbance at 260 nm (UV spec) if the UV spec or fluorescence (with oligreen) if UV spec is not sensitive enough?

--
Phillip

Hi,

Yes, I've measured them with picogreen... I guess I should have phrased the questions slightly differently...

"Can anyone confirm that this will work, having actually switched the adapters after dilution and produced an experimentally tested library".

I expect that it will be fine, but whenever I modify the protocols I always like to check whether anyone else has already tried it before I blow a few thousand dollars finding out the hard way :-)

Cheers,

Scott.

I guess picogreen should not work well. It is a double stranded fluor that fluoresces very weakly in the presence of single stranded molecules. Hence it is not confounded much by RNA contamination of a DNA sample. For short oligonucleotides anneal over a short stretch I don't know you would get much signal.

But if your concentration results were fairly linear over a set of dilutions, I guess you will be okay.

Anyway, no, we have not tried it ourselves. Please let us know how it turns out.

--
Phillip

Hi,

Yes, I realise PG detects primarily dsDNA, but I think the adapters have sufficiently large double stranded regions to work with picogreen. We really only need a relative concentration rather than an abolute concentration.

It appears that they're approximately 60 fold more dilute in the RNA-seq kit than in the genomic DNA kit.

We diluted the DNA adapters 1 in 50 and the preps appear to have worked well.

Cheers,

Scott.