Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas

Abstract

Laser capture microdissection (LCM) is a powerful method to isolate specific populations of cells for subsequent analysis such as gene expression profiling, for example, microarrays or ribonucleic (RNA)-Seq. This technique has been applied to frozen as well as formalin-fixed, paraffin-embedded (FFPE) specimens with variable outcomes regarding quality and quantity of extracted RNA. The goal of the study was to develop the methods to isolate high-quality RNA from islets of Langerhans and pancreatic duct glands (PDG) isolated by LCM. We report an optimized protocol for frozen sections to minimize RNA degradation and maximize recovery of expected transcripts from the samples using quantitative real-time polymerase chain reaction (RT-PCR) by adding RNase inhibitors at multiple steps during the experiment. This technique reproducibly delivered intact RNA (RIN values 6–7). Using quantitative RT-PCR, the expected profiles of insulin, glucagon, mucin6 (Muc6), and cytokeratin-19 (CK-19) mRNA in PDGs and pancreatic islets were detected. The described experimental protocol for frozen pancreas tissue might also be useful for other tissues with moderate to high levels of intrinsic ribonuclease (RNase) activity.

Mapping Molecular Datasets Back to the Brain Regions They are Extracted from: Remembering the Native Countries of Hypothalamic Expatriates and Refugees

“…While these values are in the higher range it does indicate some degree of degradation. These findings are also consistent to the RIN values (6.2) reported from tissue collected from patients with oral cancer that was stored for ~48 h at –80°C [466], as well as RIN values (6–7) reported from pancreatic tissue collected from rats and humans [50]. They are also consistent with RIN values (6.6–7.6) reported for hypothalamic tissue sampled using LCM from the supraoptic (SO) nucleus; the LCM was performed within one month following tissue sectioning and storage of the slide-mounted sections at –80°C [203].…”

In the setting of β-cell stress, the pancreatic duct gland transcriptome shows characteristics of an activated regenerative response

“…All experimental procedures were approved by the UCLA Institutional Animal Care and Use Committee. HIP rats develop overt diabetes between 9 and 12 months of age with islet pathology similar to humans with type 2 diabetes, specifically 6 ongoing beta cell apoptosis with a progressive defect in beta cell mass (5). HIP rats used for this study were ~6 months of age, and so prediabetic with ongoing beta cell apoptosis but without the confounding secondary actions of hyperglycemia on gene expression (15).…”

“…On the day of study, animals were anesthetized by inhalation of isoflurane (Abbott Laboratories, Chicago, IL). Rat pancreas was rapidly dissected, divided into two portions (head and body of pancreas, and tail of pancreas), and cryopreserved in Optimal Cutting Temperature (OCT) compound (7). [10][11][12][13][14][15] Sections were cut from the head of the pancreas for each LCM experiment.…”

“…After identification of the compartment by microscopy, laser dissection is used to procure a sample of the cells of interest to permit subsequent gene expression and/or proteome profiling (11,21). In the present study we employed that approach to procure high quality RNA by a protocol validated for PDGs (7). We then characterized the transcriptional profile of these samples using RNA sequencing (RNAseq) to establish the molecular identity of the PDG compartment in an unbiased manner.…”

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

“…Whole tumour biopsies are typically used in conventional techniques of RNA-Seq, limiting the possibility of further differentiation of specific gene expression in various tumour cell types. Furthermore, conventional methods typically require the capture of a significant number of cells or numerous cycles of RNA amplification to generate ample quantities of RNA for downstream experiments, which may inadvertently introduce artifacts especially when initial RNA quantities are small [26][27][28] . In order to precisely characterize genomic aberrations in a tumour population, it is integral to acquire pure genetic material from cancerous cells with no or minimal contamination of neighbouring cells.…”