Abstract
Advances in high-throughput sequencing technologies have enabled extensive studies of freshwater biofilms and significant breakthroughs in biofilm meta-omics. To date, however, no standardized protocols have been developed for the effective isolation of RNA from freshwater benthic biofilms. In this study, we compared column-based kit RNA extraction with five RNAzol-based extractions, differentiated by various protocol modifications. The RNA products were then evaluated to determine their integrity, purity and yield and were subjected to meta-transcriptomic sequencing and analysis. Significant discrepancies in the relative abundance of active communities and structures of eukaryotic, bacterial, archaebacterial, and viral communities were observed as direct outcomes of the tested RNA extraction methods. The column isolation-based group was characterized by the highest relative abundance of Archaea and Eukaryota, while the organic isolation-based groups commonly had the highest relative abundances of Prokaryota (bacteria). Kit extraction methods provided the best outcomes in terms of high-quality RNA yield and integrity. However, these methods were deemed questionable for studies of active bacterial communities and may contribute a significant degree of bias to the interpretation of downstream meta-transcriptomic analyses.
Highlights
In freshwater environments, benthic biofilms can be considered “cities of microorganisms” that attach to abiotic surfaces and embed into extracellular polymeric substances
Reliable high-quality total nucleic acid (RNA) extraction is a prerequisite for recovering messenger reliable high-quality total nucleic acid (RNA) for gene expression studies based on next-generation sequencing, quantitative reverse transcription polymerase chain reaction or microarrays, as low-quality total RNA may strongly compromise the experimental results and waste time, labor, and resources (Fleige and Pfaffl, 2006; Riedmaier et al, 2010)
The bacterial community profiles in freshwater benthic biofilms were assessed to provide a reference for this study
Summary
Benthic biofilms can be considered “cities of microorganisms” that attach to abiotic surfaces and embed into extracellular polymeric substances. These freshwater biofilms have been identified as major sites of carbon and nutrient cycling (Lear et al, 2012) and are thought to play an important role in the maintenance and monitoring of freshwater health. The study of biofilm community structures and their functional attributes require a proper nucleic acid extraction, regardless of the sequencing platform or molecular analysis method. A plethora of nucleic acid extraction methods exist, ranging from commercial kits to protocols involving homemade reagents. These methods include diverse mechanical, chemical, and/or enzymatic lysis methods and diverse post-extraction purification methods. Several methods to extract total RNA from environmental samples have been described in the literature (Hurt et al, 2001; Sessitsch et al, 2002; Wang et al, 2012), few published studies have tested and compared these methods when applied to environmental biofilm samples
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
