Show simple item record

dc.contributor.authorFarris, Shannonen
dc.contributor.authorWang, Yuen
dc.contributor.authorWard, James M.en
dc.contributor.authorDudek, Serena M.en
dc.date.accessioned2019-06-03T21:02:59Zen
dc.date.available2019-06-03T21:02:59Zen
dc.date.issued2017-06-17en
dc.identifier.citationFarris S, Wang Y, Ward JM and Dudek SM (2017) Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq. Front. Mol. Neurosci. 10:185. doi: 10.3389/fnmol.2017.00185en
dc.identifier.urihttp://hdl.handle.net/10919/89709en
dc.description.abstractObtaining high quality RNA from complex biological tissues, such as the brain, is needed for establishing high-fidelity cell-type specific transcriptomes. Although combining genetic labeling techniques with laser capture microdissection (LCM) is generally sufficient, concerns over RNA degradation and limited yields call into question results of many sequencing studies. Here we set out to address both of these issues by: (1) developing a fluorescence-assisted LCM protocol that yields high quality RNA from fresh-frozen tissues; and (2) determining a suitable RNA-Seq library generation method for limited amounts of RNA (1–5 ng total RNA). The latter focused on comparing commercially available kits able to produce libraries of sufficient concentration and complexity while limiting PCR amplification biases. We find that high quality RNA (RNA integrity number, RIN, >9) of sufficient concentration can be isolated from lasercaptured material from thinly-sectioned tissues when digestion time and temperature are minimized. Furthermore, we found that library generation approaches that retain ribosomal RNA (rRNA) through cDNA library generation required fewer cycles of PCR, minimizing bias in the resulting libraries. Lastly, end stage depletion of rRNA prior to sequencing enriches for target RNAs, thereby increasing read depth and level of gene detection while decreasing sequencing costs. Here we describe our protocol for generating robust RNA-Seq libraries from laser-captured tissue and demonstrate that with this method, we obtain samples with RNA quality superior to the current standard in the LCM field, and show that low-input RNA-Seq kits that minimize PCR bias produce high fidelity sequencing metrics with less variability compared to current practices.en
dc.description.sponsorshipThis work was supported by the Intramural Research Program of the U.S. National Institutes of Health, National Institute of Environmental Health Sciences [Z01 ES100221 to SMD].en
dc.format.extent13 pagesen
dc.format.mimetypeapplication/pdfen
dc.language.isoenen
dc.publisherFrontiersen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectRNA-Seqen
dc.subjectlaser-capture microdissectionen
dc.subjectRNA qualityen
dc.subjectlow-inputen
dc.subjecthippocampusen
dc.subjecttranscriptomeen
dc.titleOptimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seqen
dc.typeArticle - Refereeden
dc.title.serialFrontiers in Molecular Neuroscienceen
dc.identifier.doihttps://doi.org/10.3389/fnmol.2017.00185en
dc.identifier.volume10en
dc.type.dcmitypeTexten


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record

Creative Commons Attribution 4.0 International
License: Creative Commons Attribution 4.0 International