Problems with cDNA 1st strand synthesis

[Spanading]

Hi Seq peps!

I am hoping someone has done this before and knows the answer. I am using one of the targeted expression kits from Illumina to profile a selection of genes using a 48 target panel. However, I have just perfromed the cDNA synthesis step at this stage and wanted to check the quality of the cDNA. Having spoken to the people at Agilent as to the best Bioanalyser chip to use, I ran a selection of the samples I wanted to run on an RNA pico chip. The results are puzzeling however. In the inital RNA samples,while the quality was not optimal, I did at least seem ribosomal peaks in all samples when run on the pico chips. and there was a broad spread of peak sizes (although some samples were a bit on the low side). However, when running 1µl of the cDNA synthesis mix I can only see one large peak at around 25bp which then tapers off to background at about 200-300bp. I do know the lady at Agilent said the chip might not run correctly due to the presence of cDNA but the strange profile and the lack of any ribosomal peak does concern me, (unless anyone knows if Illumina are combining RNA fragmentation and ribosomal RNA depletion in the cDNA synthesis step, which might explain the odd 25C incubation step that i had assumed was for the binding of the random primers only). Any help would be appreciated so I can assess whether to continue with these samples.

Cheers

Spencer

[Spanading]

OK can people sanity check this idea that myself and a couple of colleagues came up with please? We made a couple of assumptions along the way, so the argument goes. If the Illumina targetted expression kit primes the reverse transcription reaction using an oligo dT then presumably the ribosomal RNA would not be targetted, and as the sample is diluted by at least a factor of 2 during the reverse transcription step then this may well make the ribosomal peaks more difficult to see. Further the big peak at 20 bps or so is probably the excess of primer and maybe causing the scale to be too large to see the peaks of the ribosomal genes that are there. As the mRNA is species will vary in size this would explain the increased background. Alternatively (and I hope not the case) it could just be all the RNA is degraded and this is reflected by the large peak at 20 or so bp that then tapers off on its right hand side.

[SunPenguin]

You could try to quantify the cDNA (I assume you mean first strand cDNA, ssDNA), but I honestly think it's a lost cause.

I presume you cleaned your cDNA using some column/SPRI, but even then, cDNA won't always run as expected on BioA.

I would just bioA the RNA, then do WTA on the cDNA and check the resulting dsDNA on a regular chip afterwards, and see if your amplification is good. If degradation is an issue, you'll probably see poor results