Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

Summary

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

Abstract

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

Introduction

Technological advances in studying tissue specimens have improved understanding of the state of health and disease in various organs. Such advances have underscored that pathology can start in limited regions or in specific cell types, yet have important implications on the entire organ. Therefore, in the current era of personalized medicine, it is important to understand the biology at both the cell and regional level and not only globally¹. This is particularly true in the kidney, which is composed of various specialized cells and structures that differentially initiate and/or respond to pathological stress. The pathogenesis of various types ....

Protocol

The study was approved for use by the Institutional Review Board (IRB) at Indiana University.

NOTE: Use this protocol with kidney nephrectomy tissue (up to 2 cm in both the X and Y dimensions) preserved in the Optimal Cutting Temperature (OCT) compound and stored at -80 °C. Perform all work in a manner that limits RNA contamination, use clean disposable gloves and a face mask. Ensure the cleanliness of all surfaces. The equipment for which this protocol was optimized is a laser microdissection system featuring pulsed UV laser.

1. Cryosectioning

1. Expose 1.2 µm LMD PPS-membrane (poly(p-phenylen....

Results

Samples

We present data from nine reference nephrectomies (3 specimens obtained at Indiana University and 6 specimens obtained through Kidney Precision Medicine Project), utilizing the rapid fluorescence staining protocol to isolate kidney nephron segments and interstitial areas. The sections utilized in this process were obtained from deceased kidney donors or unaffected tumor nephrectomies. These samples did not have pathologic evidence of disease as visualized in the H&.......

Discussion

LMD based transcriptomics is a useful technology that anchors gene expression to specific areas within the tissue. The basis of this technology and its potential application in the kidney has been described previously⁸. However, optimization, modernization and streamlining of fluorescence-based dissection specifically aimed at high accuracy dissection for downstream RNA sequencing is less ubiquitous. Because this methodology is spatially grounded within the tissue, it has the potential to reveal n.......

Materials

Name	Company	Catalog Number	Comments
Acetone	Sigma-Aldrich	270725-1L
AMPure Beads	Beckman Coulter	A63880
Bioanalyzer	Agilent	2100
BSA	VWR	0332-100G
DAPI	ThermoFisher	62248
Desiccant Cartridge	Bel-Art	F42046-0000
DNAse	Qiagen	79254	RDD buffer is included in the pakage
Laser Microdissection Microscope	Leica	LMD6500
Megalin/LRP2 Antibody	Abcam	ab76969	Directly conjugated to Alexa Fluor 568
Microcentrifuge tubes	ThermoFisher	AB-0350
Microscope camera	Leica	DFC700T
PBS (RNAse Free)	VWR	K812-500ML
Phalloidin (Oregon Green 488)	ThermoFisher	O7466
PicoPure RNA Isolation Kit	Applied Biosystems	KIT0204
PPS-membrane slides	Leica	11505268
qPCR Human Reference Total RNA 25 µg	Takara Clontech	636690
RNA 6000 Eukaryote Total RNA Pico Chip	Agilent	5067-1513
RNAse Away	ThermoFisher	7000
RNAse Inhibitor	ThermoFisher	AM2696
Sequencer (HiSeq or NovaSeq)	Illumina	NA
SMARTer Stranded Total RNAseq Pico Input v2	Takara Clontech	634411
Tamm-Horsfall Protein Antibody	R&D Systems	AF5144	Directly conjugated to Alexa Fluor 546
Tissue-Tek® O.C.T. Compound	Sakura	4583