Dear all,
I'm currently preparing small RNA library for Illumina sequencing. I have two samples: one is wild-type cells, and the other is mutant cells in which one of the miRNA biogenesis factor had been knocked out. As expected, these KO cells are virtually devoid of canonical miRNAs, as determined by northern blotting (although few miRNAs whose biogenesis occur through alternate pathways are still expressed).
I started library prep with an equal amount (10 μg) of total RNA from each cells, size-fractionated on denaturing PAGE to enrich 18-30 nt RNAs, ligated adaptors at both ends and performed RT-PCR. Preliminary PCR result suggests more than 10-fold differences in the amount of PCR product after equal number of thermal cycling.
I initially tried to keep the number of cycles identical between the two samples. However, I realized that it is very hard to determine the optimal cycle number which is not too low to get decent amplification from KO sample and is not too high to avoid overamplification and accumulation of PCR artifacts from WT sample.
Stuck in this situation, I searched literatures and found a few studies reporting that, unlike in other library prep, PCR introduces negligible bias during small RNA library preparation (one is from Tom Tuschl lab, 2011 RNA). So I wonder if I can give different number of cycles (differing more than 4) to each sample to get similar amount of PCR product.
Hi Dooyoung, I wouldn't recommend using different pcr cycles. Use at the higher end of recommended cycles for both groups if you are not getting enough yield. But you don't need very much library to sequence. I know of a similar study using Illumina strategy with Dicer KO where they observed miRNA at RPKM of 5-10 and saw significant differences in those low expressed miRNA as expected since it's a major regulator of miRNA biogenesis. Using different cycles would introduce bias and comparisons between the groups wouldn't be valid. We combined barcoded small RNA libraries after pcr and size selected on gel. The bioanalyzer concentration (within library size region) was used to pool all libraries. This way you are not limited by final yield at pcr stage, which would be low. We used Illumina kit and followed their guidelines.
Dear Pabalu, I really apprecitate your kind reply. I'm curious about the multiplexing process you mentioned; you first run each library on the bioanalyzer, estimate individual region molarity, pool libraries together at equimolar ratio, then gel purify, right?
I would suggest to you to prepare your library from total RNA (no size enrichment of miRNA), ligate adapters, RT-PCR amplify then size select your miRNAs on a gel using the total size of miRNA+adapters.
Hi Dooyoung, make a region of miRNA + adapter (with Illumina kit, it's ~130-170nt) in the bioanalyzer, get molarity, pool all libraries equimolar, run the pool on a PAGE gel to size select, gel extract using a crush and soak protocol. It worked great for us. I agree with Zineb about starting with total RNA. That's what we did as well. But we ran our total RNA on a nano chip and on a small RNA chip, quantified % miRNA = mass of miRNA (10-40nt) from small RNA chip/mass of Total RNA from nano chip. If your miRNA % > 0.5, no enrichment of small RNA is needed.
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