Abstract
Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1–10 ng of intact total RNA and 100–500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues.
Highlights
Ribosomal RNAs constitute >90% of the total RNA mass within cells [1,2]
Previous studies have shown that Ribosomal RNA (rRNA) depletion protocols are more robust than poly (A)based protocols for use in applications with lower total RNA amounts or degraded RNA [5,6,7,8,9,10,11,12,13,14]
We performed analyses to identify an optimal method for such applications, comparing selected commercially available rRNA depletion protocols
Summary
Ribosomal RNAs (rRNAs) constitute >90% of the total RNA mass within cells [1,2]. To enhance the sensitivity of RNA-seq to rare mRNA transcripts, methods for either enriching for mRNAs or depletion of rRNAs have been developed. Alternative strategies that address this bias are based on the specific removal of rRNAs [5,6,7] These strategies have the added potential advantage of capturing non-ribosomal transcripts that lack polyadenylated tails. A widely adopted commercial kit, illustrative of the type of strategy used in rRNA depletion-based protocols, is the Ribo-Zero Gold kit (Illumina). This protocol uses negative selection of rRNAs via magnetic bead-based affinity purification [5]. We compare two available rRNA depletion kits using intact and FFPE RNA samples across a range of total RNA input amounts.
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