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Old 10-20-2017, 08:26 AM   #1
valopes
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Default If I have a miRNA purified sample, can I use TruSeq Stranded mRNA LT for library prep

Hello all,
I've been looking for papers but I am still in doubt If could use the kit that I have in the lab, TruSeq® Stranded mRNA LT (Illumina), for library prep of my miRNA samples. I already did the RNA extraction, using miRNeasy Mini Kit (Qiagen), and enrichment of miRNA and RNAs <200 nucleotides, using miRNeasy Mini Kit (Qiagen). Does anyone that knows better about this, could please tell me?
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Old 10-20-2017, 09:39 AM   #2
pmiguel
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TruSeq® Stranded mRNA LT (Illumina) is designed to fragment long RNAs using heat and divalent cations and then prime 1st and 2nd strand synthesis using hexamer primers.

miRNAs are a specific type of very short RNA -- typically around 21 nucleotides long. They are somewhat unusual in having being created by an RNAse that leaves a 5'-phosphate and 3'-hydroxyl. Which means both ends are natively substrates for T4-RNA-ligase-1. So, nearly universally, library kits for miRNA sequencing directly ligate adapters onto the ends of these very short RNA fragments. Then first and 2nd cDNA synthesis is undertaken, using the adapters to prime them.

The result is that many inserts are just a single, full-length, processed miRNA.

Whereas, if you just fragment a pool of RNA that includes miRNAs then anneal hexamers to them (positioned randomly) to prime cDNA synthesis, your chances of getting a full length insert are low.

So, I would not recommend that kit for construction of a miRNA library. Instead consider a kit or a methodology designed for miRNA.

--
Phillip
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