Targeted Transcriptional Profiling of Microdissected Biopsy Specimens Representing Early Colonic Neoplasia

Mo, Allen; Jackson, Sara L.; Devers, Thomas J.; Rosenberg, Daniel W.

doi:10.1002/jcb.25644

Cited by 3 publications

(3 citation statements)

References 19 publications

(25 reference statements)

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“…In our system, the shorter incubation time in the QIAGEN kit did not significantly affect the RNA yield/area, suggesting that longer lysis incubation times negatively affect RNA quality without much gain in RNA yield. Interestingly, when comparing previous LCM articles that used either PicoPure (Butler et al, 2016; Mo et al, 2016) or QIAGEN (Wang et al, 2009; Cummings et al, 2011) kits for RNA isolation, those that used the QIAGEN kit reported higher RIN values compared to those that used the PicoPure kit, confirming that our findings are robust and translatable to many different types of samples. One study (Mo et al, 2016), also compared RNA isolated using the PicoPure kit with another RNA extraction kit, RNAqueous-Micro Kit, that reportedly had faster extraction times at a lower temperature and a smaller elution volume.…”

Section: Discussionsupporting

confidence: 80%

Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

Farris

Wang

Ward

et al. 2017

Front. Mol. Neurosci.

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Obtaining 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 laser-captured 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.

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Section: Discussionsupporting

confidence: 80%

Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

Farris

Wang

Ward

et al. 2017

Front. Mol. Neurosci.

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“…The ability to generate tissue-specific expression profiles can significantly expand our knowledge on the pathophysiological mechanisms of different diseases, providing information on functional elements and molecular constituents of the tissues and identifying new diagnostic and therapeutic biomarkers [ 1 ]. High-throughput gene expression profiling through next generation sequencing (NGS) has several advantages over microarray technology, such as single-nucleotide resolution and the possibility to study the entire RNA content of a single cell, cell populations or tissues starting from nanograms quantities of input material [ 2 , 3 ]. Moreover, by assaying millions of transcripts in RNA sequencing data, it is also possible to discover gene fusion, alternative splicing and novel isoforms expanding the complexity of gene expression field not possible with microarray technology [ 1 , 2 ].…”

Section: Introductionmentioning

confidence: 99%

Laser capture microdissection for transcriptomic profiles in human skin biopsies

et al. 2018

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BackgroundThe acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-frozen human specimen by laser capture microdissection, the abundant presence of skin nucleases and RNA instability remain relevant methodological challenges. We developed and optimized a protocol to extract RNA from layers of human skin biopsies and to provide satisfactory quality and amount of mRNA sequencing data.ResultsThe protocol includes steps of collection, embedding, freezing, histological coloration and relative optimization to preserve RNA extracted from specific components of fresh-frozen human skin biopsy of 14 subjects. Optimization of the protocol includes a preservation step in RNALater® Solution, the control of specimen temperature, the use of RNase Inhibitors and the time reduction of the staining procedure. The quality of extracted RNA was measured using the percentage of fragments longer than 200 nucleotides (DV200), a more suitable measurement for successful library preparation than the RNA Integrity Number (RIN). RNA was then enriched using the TruSeq® RNA Access Library Prep Kit (Illumina®) and sequenced on HiSeq® 2500 platform (Illumina®). Quality control on RNA sequencing data was adequate to get reliable data for downstream analysis.ConclusionsThe described implemented and optimized protocol can be used for generating transcriptomics data on skin tissues, and it is potentially applicable to other tissues. It can be extended to multicenter studies, due to the introduction of an initial step of preservation of the specimen that allowed the shipment of biological samples.Electronic supplementary materialThe online version of this article (10.1186/s12867-018-0108-5) contains supplementary material, which is available to authorized users.

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“…Additionally, LCM enables the isolation of homogeneous populations of cells from histologically complex tissues sections 31 . This approach results in satisfactory quality and quantity of RNA for subsequent experiments, such as microarray analyses 32,33 . It is a valuable technique that permits extraction of deeply embedded cells from tissues and has multiple advantages compared to conventional techniques.…”

Section: Discussionmentioning

confidence: 99%

An Optimised Protocol Harnessing Laser Capture Microdissection for Transcriptomic Analysis on Matched Primary and Metastatic Colorectal Tumours

Ong

Tan

Lim

et al. 2020

Sci Rep

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Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework's potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution. High-throughput next-generation sequencing (NGS) is a powerful strategy to study cancer progression at the molecular level, where ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity 1. By comparing the transcriptomes, differential expression of genes in discrete cell populations can be easily identified. This approach has emerged as a useful tool for characterising the transcriptomes and molecular signatures of tissues-of-interest via RNA or protein profiling, which may reflect their functionality 2. In recent years, large genomic consortiums such as The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC) have been established 1. With these publically available large-scale integrated data, much effort have been placed into identifying putative prognostic biomarkers using NGS, however, few have made it to clinical practice 3. A plausible reason for this could be due to the

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Targeted Transcriptional Profiling of Microdissected Biopsy Specimens Representing Early Colonic Neoplasia

Cited by 3 publications

References 19 publications

Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

Laser capture microdissection for transcriptomic profiles in human skin biopsies

An Optimised Protocol Harnessing Laser Capture Microdissection for Transcriptomic Analysis on Matched Primary and Metastatic Colorectal Tumours

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