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
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
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