mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets

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.

Streamlined and sensitive mono- and diribosome profiling in yeast and human cells

“…Given the relatively limited rRNA digestion by P1 nuclease ( Figure 2e ), we wanted to test whether the technically demanding gel-based RPF size selection could be avoided. We replaced gel-based RPF size selection with mirRICH small RNA enrichment, which relies on differential resuspension of small (< 200-300 nt) versus large RNAs from precipitated RNA [39]. We performed size selection only after OTTR cDNA synthesis, enriching library cDNAs with either monosome-size (30 – 45 nt) and/or disome-size (50 – 80 nt) inserts.…”

“…The ribosome pellets were first completely resuspended in a 60 µl polysome buffer and then combined with 600 µL TRIzol and incubated for 10 minutes. The sample was then split in half, with 330 µL purified by Direct-Zol and 330 µl by mirRICH [39]. Direct-Zol RNA was eluted in 100 µL, 10 µL 3 M sodium acetate pH 5.5 and 300 µL of 100% ethanol was added, and RNA was precipitated at -80 °C for 3 hours.…”

Denitrification characterization of dissolved oxygen microprofiles in lake surface sediment through analyzing abundance, expression, community composition and enzymatic activities of denitrifier functional genes

“…Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions. Total RNA was extracted using the acid guanidium thiocyanate-phenol–chloroform (AGPC) method (Choi et al 2018). After extraction, RNA was reversely transcribed into cDNA using Superscript II reverse transcriptase (Life Technologies Corporation, USA) in accordance with manufacturer’s instructions.…”

High-quality RNA isolation from pigment-rich Dendrobium flowers

“…An incubation time of 45 min was found to result in a higher yield and good quality of RNA (Supplementary Table S3). Another contributing factor for low pellet solubility might be the use of isopropanol in Methods 1 and 3, because isopropanol promotes the co-precipitation of salts (Choi et al 2018). It is also difficult to completely remove isopropanol from samples because of low volatility, compared to ethanol, which further deteriorates the quality of RNA (Surzycki 2012).…”