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We are preparing RNA-Seq libraries for Illumina HiSeq 3000. However, a few of the libraries are low in concentration (<5 nM, tested on Agilent Bioanalyzer High Sensitivity DNA chip) and we can not go back for RNA isolation. Can we take aliquot of 2 ul from the library and re-amplify for 5 cycles (and bead purify) to ensure proper yield? And can we use this data for following analysis (compare gene expression levels)?
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Hi Jin1,
I assume you will be pooling multiple libraries? ; thus, the molarity of the final pool will have to meet the requirements of your sequencing service -- not the individual libraries.
If you also have higher concentrated libraries it is very likely that the final concentration will be no problem.
I would very much avoid amplifying a subset of the libraries in additional PCR reactions and cleanups - this likely could introduce technical variation. I would rather amplify all if absolutely needed. -
Hi Luc,
Thanks for your reply! The re-amplification is only for one or two samples (out of hundred; and are from different biological replicates) which failed to get enough yield.Comment
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The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
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