Journal: Scientific Reports
Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes
Figure Lengend Snippet: Quantitative and qualitative comparison of RNA recovered following rRNA depletion. A total of 4 μg of DNAse-treated RNA, isolated form 3-day-old P. aeruginosa PAO1 biofilms, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using Qubit fluorimetric quantitation with the Qubit RNA HS Assay Kit. Yields are reported as total RNA recovered ( A ) and as percentage of the input RNA ( B ). The RNA samples were also assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( C ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( D ) MICROBExpress, ( E ) RiboMinus, or ( F ) Ribo-Zero kits are shown. Dashed lines indicate peaks corresponding to 16S and 23S RNA traces, which were detected in total RNA, MICROBExpress, and RiboMins samples. ( G ) The area of 16S and 23S rRNA peaks as percent of the total detected RNA was estimated, as determined using the 2100 Expert Software. RFU, relative fluorescence units. Experiments were repeated using three biological replicates.
Article Snippet: RNA and DNA quantity and quality assessment Where indicated, RNA or DNA was quantified using Qubit RNA HS Assay Kit and Qubit dsDNA HS Assay Kit (Life Technologies), respectively, on the Qubit 2.0 Fluorometer (Life Technologies).
Techniques: Isolation, Quantitation Assay, RNA HS Assay, Software, Fluorescence