RNAsnap™: a rapid, quantitative and inexpensive, method for isolating total RNA from bacteria

Abstract

RNAsnap™ is a simple and novel method that recovers all intracellular RNA quantitatively (>99%), faster (<15 min) and less expensively (∼3 cents/sample) than any of the currently available RNA isolation methods. In fact, none of the bacterial RNA isolation methods, including the commercial kits, are effective in recovering all species of intracellular RNAs (76-5700 nt) with equal efficiency, which can lead to biased results in genome-wide studies involving microarray or RNAseq analysis. The RNAsnap™ procedure yields ∼60 µg of RNA from 10(8) Escherichia coli cells that can be used directly for northern analysis without any further purification. Based on a comparative analysis of specific transcripts ranging in size from 76 to 5700 nt, the RNAsnap™ method provided the most accurate measure of the relative amounts of the various intracellular RNAs. Furthermore, the RNAsnap™ RNA was successfully used in enzymatic reactions such as RNA ligation, reverse transcription, primer extension and reverse transcriptase-polymerase chain reaction, following sodium acetate/ethanol precipitation. The RNAsnap™ method can be used to isolate RNA from a wide range of Gram-negative and Gram-positive bacteria as well as yeast.

Figure 1.

Quality assessment of RNA samples isolated by each method. (A) A representative composite bioanalyzer digital gel image using two technical replicates of each of the RNA extraction method tested (see ‘Materials and Methods’ section). (B) A representative composite image of technical replicates of 250 ng of total RNA (based on A₂₆₀) from each RNA extraction method electrophoresed on a 1.2% agarose–0.5× TBE gel and stained with ethidium bromide.

Figure 2.

Northern analysis of specific RNA species using total RNA isolated by each RNA isolation method. Five micrograms of total RNA (based on A₂₆₀ and two independent technical replicates) were used for northern analysis on each of the eight specific RNAs listed on the sides of the autoradiograms along with the approximate size of each transcript. The rpsJ, adhE and ompF transcripts were separated on agarose gels while the rest of the transcripts were separated on polyacrylamide gels (see ‘Materials and Methods’ section).

Figure 3.

Comparison of RNAsnap™ and Trizol® Max™ isolated RNA in an RT–PCR experiment. RNA isolated from SK4390 (rph-1 ΔrppH) was reversed transcribed with a primer specific for the lpp mRNA (∼330 nt) and subsequently PCR amplified for either 5,10,15, or 20 cycles (see ‘Materials and Methods’ section). The amplified PCR products were run on a 2% agarose gel and quantitated using ImageQuant TL software (GE). The amount of PCR product at the end of a fixed number of cycles from RNAsnap™ isolated RNA was set at 1 and compared with the amount of product obtained using Trizol® Max™ isolated RNA. Lanes 1 and 12, Gene Ruler™ Low Range DNA Ladder (Fermentas).

Figure 4.

Primer extension analysis using RNAsnap™ isolated RNA. The primer extension was carried out as described in ‘Materials and Methods’ section. Lanes 1–4, sequencing ladder derived from rrnB operon. Leftward arrow indicates the mature 5′-terminus of 23S rRNA (wild-type, lane 5), which is missing in the absence of RNase III (rnc-14, lane 6).