Impact of RNA Isolation Protocols on RNA Detection by Northern Blotting

Abstract

We prepared total RNA from the Gram-positive soil bacterium Bacillus subtilis by different RNA extraction procedures to compare their suitability for Northern blot detection of tiny RNAs (~14-mers) or RNAs of intermediate size (100-200 nt) in terms of signal quality, intensity, and reproducibility. Our analysis included two hot phenol methods and two TRIzol extraction procedures. We found that signal intensity/detection sensitivity makes the key difference. Total RNAs prepared by the hot phenol method comprise the length spectrum from tRNAs to large ribosomal RNAs. Larger RNAs are less abundant in TRIzol preparations which instead enrich for RNAs of tRNA size and smaller. Thus, hot phenol methods are the choice for the detection of intermediate-sized and longer RNAs, whereas TRIzol extraction procedures are more suited for the detection of tiny RNAs.

Bacillus subtilis 6S-2 RNA serves as a template for short transcripts in vivo

“…To validate this trend seen in the RNA-seq data, we performed 6S-1 pRNA-specific Northern blot analyses using total RNA extracted from the wt and mutant strains. However, with about 10 independent RNA extracts prepared from wt and ΔbsrB bacteria in parallel, either using the hot phenol or TRIzol method (Damm et al 2015), no consistent results were obtained. With RNA prepared by the hot phenol method (as done in the RNAseq experiment), we could see higher pRNA levels for the ΔbsrB relative to the wt library in most experiments, but not all.…”

“…With the following modifications we were able to detect 6S-2 pRNAs (Fig. 2): We used RNA/LNA mixmer probes with a high LNA content (up to 10 out of 14 nt), a double digoxigenin label at the 5 ′ -and 3 ′ -end (to increase signal intensity), and applied the TRIzol RNA extraction method to enrich for small RNAs (Damm et al 2015). In Northern blots of the type shown in Figure 2B, we detected signals for 6S-2 pRNAs in the range of ∼14-25 nt for RNA preparations from wt and ΔbsrA bacteria, but not for those originating from ΔbsrB and ΔbsrAB bacteria lacking the 6S-2 RNA gene.…”

“…Cellular total RNA for Northern blotting and primer extension was prepared using the TRIzol (Ambion) method. For this purpose, pelleted B. subtilis cells were resuspended in an appropriate amount of TRIzol reagent (for a pellet deriving from 50 mL LB culture, 4 mL TRIzol were used) by vortexing and incubated for 5 min on ice, followed by the procedure described in Damm et al (2015). The purity of the RNA preparation was determined by UV spectroscopy.…”

“…This was proven by three independent experimental approaches: (i) RNA-seq of cellular RNA extracts enriched for short primary transcripts using Illumina technology to obtain high read numbers, (ii) application of a Northern blot procedure with RNA fractions prepared by the TRIzol method for efficient extraction of small RNAs (Damm et al 2015), combined with a probe that confers enhanced detection sensitivity, and (iii) a novel primer extension assay which utilizes the 6S-2 pRNAs as primers on a complementary DNA template. The novel insight gained here indicates that 6S-2 RNA is a fully functional 6S RNA homolog with the ability to inhibit RNAP activity and to release RNAP from its sequestration by serving as a template for pRNA synthesis analogous to 6S-1 RNA.…”

Protein-only RNase P function in Escherichia coli: viability, processing defects and differences between PRORP isoenzymes

“…At OD 600 ∼ 1.0 (= outgrowth phase), cells were harvested and total RNA was prepared according to Method 1 (‘Extracting RNA three times with hot phenol’) described by Damm et al . (15). Library generation was performed at vertis Biotechnologie AG (Freising-Weihenstephan, Germany).…”