'Degraded' RNA profiles in Arthropoda and beyond

Abstract

The requirement for high quality/non-degraded RNA is essential for an array of molecular biology analyses. When analysing the integrity of rRNA from the barnacle Lepas anatifera (Phylum Arthropoda, Subphylum Crustacea), atypical or sub-optimal rRNA profiles that were apparently degraded were observed on a bioanalyser electropherogram. It was subsequently discovered that the rRNA was not degraded, but arose due to a 'gap deletion' (also referred to as 'hidden break') in the 28S rRNA. An apparent excision at this site caused the 28S rRNA to fragment under heat-denaturing conditions and migrate along with the 18S rRNA, superficially presenting a 'degraded' appearance. Examination of the literature showed similar observations in a small number of older studies in insects; however, reading across multiple disciplines suggests that this is a wider issue that occurs across the Animalia and beyond. The current study shows that the 28S rRNA anomaly goes far beyond insects within the Arthropoda and is widespread within this phylum. We confirm that the anomaly is associated with thermal conversion because gap-deletion patterns were observed in heat-denatured samples but not in gels with formaldehyde-denaturing.

Keywords: Bioanalyser; Degraded rNA; Denaturing; Gap deletion; Hidden break; Taxonomy.

Figure 1. Species analysed for the gap deletion.

(A) Spider Grammostola porteri; (B) Centipede Scolopendra subspinipes; and C) Barnacle Lepas anatifera.

Figure 2. Electropherogram traces for 100–200 ng of total RNA applied to an RNA Nano Chip were generated on the Agilent 2100 Bioanalyser.

RNA that appears ‘degraded’ after heat-denaturation and fails to provide a RNA Integrity Number (RIN) can generate high RINs in non-heat-denatured aliquots from the same RNA stock. RIN numbers are shown in each case for (A) Spider Grammostola porteri; (B) Centipede Scolopendra subspinipes; (C) Stalked barnacle, Order Lepadiformes, Lepas anatifera; D. Stalked barnacle, Order Lepadiformes, Dosima fascicularis E. Stalked barnacle, Order Scalpelliformes, Pollicipes pollicipes.

Figure 3. rRNA migration patterns on denaturing gel.

Non-heat-denatured samples (lanes 2–6) and denaturing gel with heat-denatured samples (3 mins at 70 °C) (lanes 8–12) using (A) 1X NorthernMax^® denaturing Gel Buffer and (B) 2X NorthernMax^® denaturing Gel Buffer. Extra bands in non-heat-denatured samples are likely to be due to secondary structures. RNA samples are from spider (lanes 2 & 8), centipede (lanes 3 & 9) and stalked barnacles (lanes 4–6 & lanes 10–12).

Figure 4. The steps involved in determining RNA quality in a species containing a ‘gap deletion’.

These include Arthropoda, most protostome animals and a scattering of other groups (-see Table 1). The schematic displays the problem with taking routine approaches in taxa with gap deletions because standard protocols specify heat-denaturation of the rRNA prior to Bioanalyser analysis. The latter measures migration and intensity of LSUs and RNA Integrity Numbers (RINs). Heat-denaturation prevents visualisation of the 28S peak in Bioanalyser electropherograms in the affected taxa, so that RNA appears ‘degraded’ and a RIN cannot be generated. The solution is to run non-heat-denatured rRNA during Bioanalyser analysis. Denaturing formaldehyde gel electrophoresis of rRNA does not appear to bring about the gap deletion and RNA subunits appear normally on these gels.

Figure 5. 28S gene sequence analysis.

(A) 28S sequence for Lepas anatifera from Illumina Hi-Seq 2000 platform; (B) The AU rich stretch of rRNA where the UAAU sequence at position 1,781 nt is likely responsible for splitting the rRNA into ∼1,780 nt and ∼2,000 nt fragments which migrate together on a heat-denaturing gel. The CGAAAGGG sequence at the 3′ end of the AU rich region (highlighted in red) is highly conserved in all 28S rRNAs; (C) Sequence alignment for 28S rDNA of centipede and barnacles upstream of the TAAT (UAAU) cleavage site (yellow highlight). The 28S rDNA sequence for spider Grammostola porteri is not available.