Effect of RNA integrity on uniquely mapped reads in RNA-Seq

Abstract

BackgroundWe examined the performance of three RNA-Sequencing library preparation protocols as a function of RNA integrity, comparing gene expressions between heat-degraded samples to their high-quality counterparts. This work is invaluable given the difficulty of obtaining high-quality RNA from tissues, particularly those from individuals with disease phenotypes.ResultsWith the integrity of total RNA being a critical parameter for RNA-Sequencing analysis, degraded RNA can heavily influence the results of gene expression profiles. We discovered that gene expression read results are influenced by RNA quality when a common library construction protocol is used. These results are based on one technical experiment from a pool of 4 neural progenitor cell lines.ConclusionsThe use of alternative protocols can allow samples with a wider range of RNA qualities to be used, facilitating the investigation of disease tissues.

Evolution of chemosensory gene families in arthropods: Insight from the first inclusive comparative transcriptome analysis across spider appendages

“…Intriguingly, this protein is essential for insects (Shukla et al. 2015) not only because of its function as hydrolase but also for its involvement in the development of the optic lobe (Chen et al. 2014).…”

Advantages of RNA‐seq compared to RNA microarrays for transcriptome profiling of anterior cruciate ligament tears

“…Nevertheless, hybridization issues with degraded clinically derived RNA, low abundance transcripts, and the availability of probes for known genes on the chip are the drawbacks of microarrays technology 3 . Like microarrays, RNA-seq also has some potential pitfalls with the use of degraded RNA samples, however, unlike microarrays, there are several protocols available that can circumvent some, if not all, of the problems associated with RNA-seq such as to remove bases adapters and overrepresented or low-quality sequences 4–6 .…”

“…3 Like microarrays, RNA-seq also has some potential pitfalls with the use of degraded RNA samples; however, unlike microarrays, there are several protocols available that can circumvent some, if not all, of the problems associated with RNA-seq such as to remove bases adapters and overrepresented or low-quality sequences. [4][5][6] RNA-seq utilizes high throughput sequencing technology to directly sequence gene transcripts and is emerging as an alternative for whole-genome transcriptome profiling. 7 RNA-seq has considerable advantages for examining transcriptome profile structure such as the detection of novel transcripts and splice junctions, although it does pose novel algorithmic and logistical challenges for data analysis and storage.…”

Purification of high-quality RNA from synthetic polyethylene glycol-based hydrogels

“…Both approaches require purification of high quality RNA from cells or tissues. However, the extraction of RNA from cells encapsulated in PEG gels results in mostly degraded RNA (low RNA integrity number (RIN)) [4, 5]. …”

“…Initially, the cellular RNA was isolated with a combination of TRIzol reagent (Life Technologies) extraction and column purification as described previously [6] (see Figure 1 for details). However, this standard procedure failed to provide high quality RNA with RIN > 7 suitable for microarray analysis or preparation of RNA-seq libraries [5] (Figure 1). Ribonuclease activity is a common cause of degradation of cellular RNAs.…”