Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

Summary

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

Abstract

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

Introduction

This method is designed to obtain high-quality RNA samples of enteric ganglia from human intestinal tissue using laser capture microdissection (LCM). The protocol described here has been optimized to provide sufficient RNA quality and yields for RNA sequencing (RNA-seq) and is intended to be used with freshly-resected, unfixed, flash-frozen human intestinal tissue.

Functional gastrointestinal and gut motility disorders affect one of every four people in the United States. The enteric nervous system (ENS), also referred to as the second brain¹, is often at the center of these disorders, as it plays a crucial role in g....

Protocol

All protocols described here have been approved by the Vanderbilt University Institutional Review Board (IRB).

1. Preparation Prior to Tissue Arrival

1. Obtain proper IRB approval and coordinate with human organ donation agencies to obtain freshly-resected, unfixed intestinal tissue from donors that meet all research criteria required for the study.
   ​Note: This protocol can be adapted for use with mice and other rodent models.
   1. Immediately upon resection of each intestinal segment, thoroughly flush the lumen with chilled PBS or tissue-storage medium to remove residual luminal material or feces.
   2. Af....

Results

We have made several improvements to existing protocols that enable the relatively rapid collection of enteric ganglia from human intestinal samples using LCM, meeting the standards for RNA-Seq. First, we optimized the rapid freezing of intestinal tissue segments in large base molds placed at the surface of a slurry of dry ice in 2-MB (Figure 1B). The best success during cryosectioning and subsequent LCM was obtained by laying intestinal segm.......

Discussion

This procedure enables the efficient collection of numerous enteric ganglia as a source to derive RNA for RNA-seq. Here, we have accelerated the processes outlined in existing protocols while maximally preserving RNA integrity. As all steps in this procedure are interdependent, it is important that all issues be eliminated from the onset of the study and that all samples are prepared as similarly to one another as possible, to obtain reliable RNA-seq.

From the onset of the procedure, RNA integ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% Neutral-buffered formalin	Sigma	HT501128-4L
2-Methylbutane	Fisher	O3551-4
3-mL syringe	BD	309628
50-mL conical tubes, polypropylene	Corning	05-526B
Base molds 37x24x5mm, disposable	Electron Microscopy Sciences	5025511
Belzer UW  Cold Storage Solution	Bridge to Life	N.A.
CapSure Macro LCM caps	Arcturus, ThermoFisher	LCM0211
Cling Wrap, Press’n Seal	Glad	N.A.
Cresyl Violet Acetate	Acros Organics	AC405760025
Cutting Board	Electron Microscopy Sciences	63308
Ethanol, 190-proof	Pharmco-AAPER	111000190
Ethanol, 200-proof	Pharmco-AAPER	111000200
Glass Microscope slides	Fisher	12-550-343
Gloves, extended cuff	Microflex	19010144
Gowns, surgical-disposable	Kimberly-Clark	19-088-2116
Tray, 20 L (large polypropylene sterilizing pan)	Nalgene	1335920B
Kimwipes (large)	Kimberly-Clark	34120
Kimwipes (small)	Kimberly-Clark	34133
Laser Capture Microdissection System (ArcturusXT)	ThermoFisher	N.A.
Leica Cryostat Chuck, large,  40 mm	Southeast Pathology Instrument Service	N.A.
Light Microscope	Olympus	CX43
Microscope objective (20X)	Olympus UPlanFl 20X/0.50, ∞/0.17	N.A.
Microscope objective (10X)	Olympus UPlanFl 10X/0.25, ∞/-	N.A.
Molecular Sieves	Acros Organics	AC197255000
Nuclease-free water	Ambion	AM9937
PicoPure RNA extraction kit	Applied Biosystems	12204-01
Pellet Pestle	Kimble Kontes	4621973
PEN membrane LCM slides	Arcturus, ThermoFisher	LCM022
RNase-free tubes, 1.5 mL	Ambion	AM12400
RNaseZAP	Sigma	Sigma-R2020
RNA Extraction Kit "A" (PicoPure)	Applied Biosystems	12204-01
RNA Extraction Kit "B" (RNeasy  Micro kit)	Qiagen	74004
RNA Extraction Method "C" (TRIzol)	Invitrogen	15596-026
RNA Extraction Kit "D" (RNeasy PLUS Mini kit)	Qiagen	74134
Splash Shield, disposable faceshield	Fisher	17-310
Scissors, Surgical, 14.5 cm "sharp-sharp"	Fine Science Tools	14002-14
Syringe filter, cellulose acetate (0.2 μm)	Nalgene	190-2520
Tissue Freezing Medium	General Data Healthcare	TFM-5
Xylenes	Fisher	X3P1GAL