I performed an RNA extraction using the Qiagen RNEasy Micro Kit, and my total RNA yield was lower than expected given the amount of input material. I'm considering whether to move forward with RNASeq on these samples, using a low-input kit for library prep from mRNA.
Should I be worried about what caused my yield to be lower than expected, or can I just move forward with a low-input kit? Does anyone have experience with the SMART-Seq v4 Ultra Low Input RNA Kit? If I can use 1 ng of RNA per sample, should I be concerned about PCR-induced bias?
Thanks!
I have used SMARTER kits from Clontech (not v4 though...) and they work pretty well down to sub ng amount of RNA. If the amplified cDNA is limited (that is trying to use as few PCR cycles as possible) normal fragmentation and library preparation can be difficult. Some use Nextera library prep to bypass that problem. I always assume some bias is present in all PCR based amplifications methods but one hopes that the effect is similar across samples. Note the less material you use the less complex the libraries get (fx results in fewer genes detected over X FPKM threshold etc...). Note also that sometimes DNA contamination can be a problem in those protocols so the DNA free RNA is better (fx DNAse treatment if possible).
Good luck !
thorarinn
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