Hello everyone,
I'm doing the mRNA library prep using Illumina TruSeq Stranded mRNA kit.
The kit recommends anything between 100-1000ng of total RNA for the prep that is quite a range. My samples are from Zebrafish embryos.
What is the best amount to use?
Would appreciate advice from more experienced users.
Total RNA and mRNA (ribosomal RNA depleted RNA) are two different things and that is the reason of a big range. for library prep 10-100 ng mRNA (amount) would be enough.
Thank you Abhijeet Singh . I know the difference between total and mRNA.
However, that doesn't answer my question. I would like to hear from someone who is using the kit or similar, what amount of input total RNA they use for their preps and what are cons and prons of using lower or higher amounts of total RNA.
Total RNA should not be and is generally not used for library prep. Total RNA will result in most of the sequence data with just ribosomal RNA reads.
Abhijeet Singh
TruSeq Stranded mRNA kit and few other kits start with total RNA.
The first step of the protocol is purification on Oligo dT beads that bind polyA mRNA molecules.
Here is the link to the protocol is you want to check: https://sapac.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_truseq/truseq-stranded-mrna-workflow/truseq-stranded-mrna-workflow-reference-1000000040498-00.pdf
Chapter 1 Page 1 might be particularly helpful.
For the best outcomes, you should use approximately 1000 ng/ul.This relies on your purity of RNA.
Based on my experience, I would use maximum volume 11ul as the protocol says, and do not care about the 1000ng/ul. because your RNA purity plays important role. If your sample is degraded (bioanalyzer), the concentration of RNA, dosen't matter. you will get low concentration of cDNA of your library. This is why, I just use 11ul from the beginning.
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