rRNA removal from Drosophila total RNA

We have received many inquiries from customers regarding the use of  the Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) with RNA from nonmammalian organisms. Although we have not tested this kit with a wide range of genera and species, it will be suitable for many organisms whose rDNA genes exhibit a high degree of homology to human rDNA. However, the rRNA removal efficiency will vary, depending on the degree of homology.

One of our customers, Dr. Dominik Handler at the Institute of Molecular Biotechnology GmbH, Austria, examined the use of the Ribo-Zero (H/M/R) Kit with Drosophila RNA. He compared the amount of rRNA depletion with the Ribo-Zero kit and a competitive kit by qPCR after one and two rounds of rRNA removal.

qPCR analysis of rRNA depletion (click to enlarge figure). Total RNA samples were treated  with one or two rounds of the Ribo-Zero kit (RZ) or a competitive kit (R-), and fold depletion calculated using qPCR with primers to multiple regions of the indicated rRNAs, after normalization to rp49 mRNA. Nitric oxide synthase (nos) mRNA was used as an internal control.

In Drosophila, as in many diptera, the 28S rRNA is processed into two parts (a “left” and “right” arm). After two rounds of rRNA removal, Dr. Handler observed depletion of the left arm by 31,000X and the right arm by 600X. Overall, the Ribo-Zero kit performed substantially better than the competitive kit.

Obtaining optimal RNA-Seq library quality

The new ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina-compatible) prepares directional, ligation-free RNA-Seq libraries in less than 3 hours, from rRNA-depleted or poly(A)+ RNA. We compared the quality of ScriptSeq libraries prepared using different methods to treat the total RNA.

We used Universal Human Reference RNA (UHRR), Brain Reference RNA (BrRR), and total RNA isolated from FFPE breast cancer tissue as starting material. The specified samples were treated with either the Ribo-Zero™ Kit, a competitive rRNA-removal kit (Company A), or a commercial oligo(dT)-based mRNA enrichment kit. For UHRR and BrRR, ScriptSeq libraries were prepared from 50-ng aliquots of the resulting rRNA-depleted or poly(A)-enriched RNA. For FFPE samples, the entire amount of rRNA-depleted RNA recovered from 500 ng total RNA input was used to prepare the libraries. The di-tagged cDNA reactions were amplified by PCR for either 10 cycles (UHRR and BrRR) or 12 cycles (FFPE) followed by Exo I digestion. Each RNASeq library was purified using MinElute (Qiagen) and recovered in 15 μl of Elution Buffer. Replicate reactions were pooled and examined using a Bioanalyzer (Agilent). Single-lane, 54-nt unidirectional sequencing reads were obtained for each library using an Illumina GAII sequencer, and sequence analysis was performed using Bowtie.

Summary of sequencing data from ScriptSeq™ libraries (click to enlarge figure). Libraries were prepared as described above from Universal Human Reference RNA (UHRR), Brain Reference RNA (BrRR), or RNA extracted from FFPE breast cancer tissue (FFPE). The indicated method of rRNA removal or mRNA enrichment was used.

As seen in the figure, optimal RNA-Seq results were obtained with the Ribo-Zero Kit for rRNA removal, even from substantially degraded RNA (FFPE sample).

Examining blocking lesions in ancient DNA

The characteristics of ancient DNA remain poorly understood. This is particularly true for blocking lesions (chemical alterations that cannot be bypassed by DNA polymerases). Blocking lesions prevent amplification and sequencing of affected molecules, thus limiting the analysis of DNA derived from ancient samples. Heyn et al. recently developed a new method--polymerase extension profiling (PEP)--that reveals occurrences of polymerase stalling on DNA templates. This sequencing-based technology allows detection of damage on a single-molecule level. The technique used CircLigase™ ssDNA Ligase for high-efficiency ligation of single-stranded adaptors (containing the Roche 454 A sequence) to the 3’ ends of primer-extension products.

The authors found evidence of blocking lesions in three out of four ancient samples, but no more than 40% of the molecules were affected, indicating that such modifications are far less frequent than previously thought.

Heyn, P. et al. (2010). Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA Nucleic Acids Research, 38 (16) DOI: 10.1093/nar/gkq572