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ABSTRACT
Techniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method “mirRICH.” The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgment
C.C., Y.U, S.Y, S.S J.S and S.R. conceived and designed the experiments. C.C, Y.U., C.B., P.D. S.Y. T.V.N, S.B, J.W and S.R. performed the experiments. J.S, J.M. and S.S. analyzed the data. C.C., Y.U, S.S and S.R. wrote the manuscript. All authors contributed to critically revise the manuscript for important intellectual content, and gave final approval and agree to be accountable for all aspects of the work. This work was supported by the Soonchunhyang University Research Fund, the Ministry of Science and ICT and Business Belt Program (2017K000492), the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017R1A2B4010480) and a grant from the Marine Biotechnology Program (No. 20140428) funded by the Ministry of Oceans and Fisheries, South Korea.
