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Ribosomal RNA depletion or exclusion has negligible effect on the detection of viruses in a pan viral microarray.

ABSTRACT: Pan viral DNA microarrays, which can detect known, novel and multiple viral infections, are major laboratory assets contributing to the control of infectious diseases. The large quantity of ribosomal RNA (rRNA) found in tissue samples is thought to be a major factor contributing to the comparatively lower sensitivity of detecting RNA viruses, as a sequence-independent PCR is used to amplify unknown samples for microarray analysis. This study aimed to determine whether depletion or exclusion of rRNA can improve microarray detection and simplify its analysis. Therefore, two different rRNA depletion and exclusion protocols, RiboMinus™ technology and non-rRNA binding hexanucleotides, were applied to the microarray sample processing and the outcome was compared with those of the sequence-independent amplification protocol. This study concludes that the two procedures, described to deplete or exclude rRNA, have negligible effect on the microarrays detection and analysis and might only in combination with further techniques result in a significant enhancement of sensitivity. Currently, existing protocols of random amplification and background adjustment are pertinent for the purpose of sample processing for microarray analysis.

SUBMITTER: McGowan S

PROVIDER: S-EPMC7119560 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Ribosomal RNA depletion or exclusion has negligible effect on the detection of viruses in a pan viral microarray.

McGowan Sarah S Nunez-Garcia Javier J Steinbach Falko F La Rocca Anna A Blake Damer D Dastjerdi Akbar A

Journal of virological methods 20140714

Pan viral DNA microarrays, which can detect known, novel and multiple viral infections, are major laboratory assets contributing to the control of infectious diseases. The large quantity of ribosomal RNA (rRNA) found in tissue samples is thought to be a major factor contributing to the comparatively lower sensitivity of detecting RNA viruses, as a sequence-independent PCR is used to amplify unknown samples for microarray analysis. This study aimed to determine whether depletion or exclusion of r ...[more]

PMID: 25034125

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Project description:BACKGROUND:The astounding regenerative abilities of planarian flatworms prompt steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed. RESULTS:In this study, we describe a workflow for the efficient depletion of rRNA in the planarian model species S. mediterranea. Our protocol is based on subtractive hybridization using organism-specific probes. Importantly, the designed probes also deplete rRNA of other freshwater triclad families, a fact that considerably broadens the applicability of our protocol. We tested our approach on total RNA isolated from stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consistent with poly(A) libraries. However, ribodepleted libraries revealed higher transcript levels for transposable elements and histone mRNAs that remained underrepresented in poly(A) libraries. As neoblasts experience high transposon activity this suggests that ribodepleted libraries better reflect the transcriptional dynamics of planarian stem cells. Furthermore, the presented ribodepletion procedure was successfully expanded to the removal of ribosomal RNA from the gram-negative bacterium Salmonella typhimurium. CONCLUSIONS:The ribodepletion protocol presented here ensures the efficient rRNA removal from low input total planarian RNA, which can be further processed for RNA-seq applications. Resulting libraries contain less than 2% rRNA. Moreover, for a cost-effective and efficient removal of rRNA prior to sequencing applications our procedure might be adapted to any prokaryotic or eukaryotic species of choice.

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Ribosomal RNA depletion or exclusion has negligible effect on the detection of viruses in a pan viral microarray.

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Ribosomal RNA depletion or exclusion has negligible effect on the detection of viruses in a pan viral microarray.

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