Name: application of laser microdissection to uncover regional transcriptomics in human kidney tissue
Uploaded: 2020-06-09T14:30:00
Description: Watch this Scientific Journal Video about Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue at JoVE.com

General: The authors would like to thank the investigators of the Kidney Precision Medicine Project (www.kpmp.org) for their gracious support and advice.
Funding: Support for this work was provided by the NIH/NIDDK K08DK107864 (M.T.E.); NIH/NIDDK UG3DK114923 (T.M.E., P.C.D.); R01DK099345 (T.A.S.). Research reported in this manuscript was supported by the National Institute of Diabetes and Digestive and the Kidney Diseases (NIDDK) Kidney Precision Medicine Project (KPMP), (www.kpmp.org), under award number U2CDK114886.
Data and materials availability: Data is archived in the Gene Expression Omnibus (GEO # pending)

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 &#176;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 microdisse...

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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...

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 of human kidney disease is still not well understood. Generating a methodology to study changes in gene expression in specific tubular segments, structures or areas of the interstitium in the human kidney will enhance the ability to uncover region specific changes that could inform on the pathogenesis of disease.
Human kidney biopsy specimens are a limited and precious resource. Therefore, technologies interrogating transcriptomics in kidney tissue should be optimized to economize tissue. The available methods to study transcriptomics at the cell and regional level include single cell RNA sequencing (scRNaseq), single nuclear RNaseq (snRNaseq), in situ spatial hybridization, and laser microdissection (LMD). The latter is well suited for precise isolation of regions or structures of interest within tissue sections, for downstream RNA sequencing and analysis2,3,4,5. LMD can be adopted to rely on identification of specific cell types or structures based on validated markers using fluorescence-based imaging during dissection.
The unique features of laser microdissection assisted regional transcriptomics include: 1) the preservation of the spatial context of cells and structures, which complements single cell technologies where cells are identified by expression rather than histologically; 2) the technology informs and is informed by other imaging technologies because an antibody marker defines expression signatures; 3) the ability to identify structures even when markers change in disease; 4) detection of lowly expressed transcripts in approximately 20,000 genes; and 5) remarkable tissue economy. The technology is scalable to a kidney biopsy with less than 100 &#181;m thickness of a core necessary for sufficient RNA acquisition and enables the use of archived frozen tissue, which are commonly available in large repositories or academic centers6.
In the ensuing work, we describe the regional and bulk transcriptomics technology in detail, optimized with a novel rapid fluorescence staining protocol for use with human kidney tissue. This approach improves upon classic LMD explorations because it provides separate expression data for the interstitium and nephron sub-segments as opposed to aggregate tubulointerstitial expression. Included are the quality assurance and control measures implemented to ensure rigor and reproducibility. The protocol enables visualization of cells and regions of interest, resulting in satisfactory acquisition of RNA from these isolated areas to allow downstream RNA sequencing.

<table>
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>270725-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure Beads</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>2100</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>VWR</td>
			<td>0332-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccant Cartridge</td>
			<td>Bel-Art</td>
			<td>F42046-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td>RDD buffer is included in the pakage</td>
		</tr>
		<tr>
			<td>Laser Microdissection Microscope</td>
			<td>Leica</td>
			<td>LMD6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Megalin/LRP2 Antibody</td>
			<td>Abcam</td>
			<td>ab76969</td>
			<td>Directly conjugated to Alexa Fluor 568</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>ThermoFisher</td>
			<td>AB-0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Leica</td>
			<td>DFC700T</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (RNAse Free)</td>
			<td>VWR</td>
			<td>K812-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin (Oregon Green 488)</td>
			<td>ThermoFisher</td>
			<td>O7466</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoPure RNA Isolation Kit</td>
			<td>Applied Biosystems</td>
			<td>KIT0204</td>
			<td></td>
		</tr>
		<tr>
			<td>PPS-membrane slides</td>
			<td>Leica</td>
			<td>11505268</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR Human Reference Total RNA 25 &#181;g</td>
			<td>Takara Clontech</td>
			<td>636690</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Eukaryote Total RNA Pico Chip</td>
			<td>Agilent</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Away</td>
			<td>ThermoFisher</td>
			<td>7000</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Inhibitor</td>
			<td>ThermoFisher</td>
			<td>AM2696</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencer (HiSeq or NovaSeq)</td>
			<td>Illumina</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SMARTer Stranded Total RNAseq Pico Input v2</td>
			<td>Takara Clontech</td>
			<td>634411</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamm-Horsfall Protein Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF5144</td>
			<td>Directly conjugated to Alexa Fluor 546</td>
		</tr>
		<tr>
			<td>Tissue-Tek&#174; O.C.T. Compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of laser microdissection to uncover regional transcriptomics in human kidney tissue

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.

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&#38;...

Watch this Scientific Journal Video about Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue at JoVE.com

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

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.

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and ...

Laser microdissection allows for the transcriptomic analysis of spatially defined regions within the kidney tissue samples. This enables us the unique opportunity to identify structures of interest even after disease induced changes took place. The main advantages of this methodology are its complementarity to other omics technologies, such as single cell RNA sequencing.It affords remarkable tissue economy and even allows the detection of lowly expressed transcripts. After acquiring the tissue sample, use a cryostat set to minus 20 degrees Celsius to cut the specimen to a thickness of 12 micrometers and use the slide adapter to fix the specimen to a specialized laser microdissection slide. Within 10 days of cryosectioning, mix 89.2 microliters of RNase-free 10%BSA in PBS with four microliters of FITC phalloidin, 1.5 microliters of DAPI, two microliters of the antibody of interest directly conjugated to Alexa Fluor 546, and 3.3 microliters of RNase inhibitor.Wash the slide in minus 20 degrees Celsius 100%acetone for one minute and place the slide in a humidity chamber. Wash the top of the slide two times with fresh RNase-free PBS for 30 seconds per wash, followed by two 30 second washes with 10%BSA in RNase-free PBS. After the last wash, treat the sample with the prepared antibody solution for five minutes at room temperature before washing two more times with 10%BSA in RNase-free PBS.After the second wash, air dry the slide for five minutes in a tissue culture hood before loading the slide onto the laser microdissection cutting platform. Install autoclaved 500 microliter micro-centrifuge collection tubes, appropriate for PCR work, containing 50 microliters of extraction buffer from an RNA isolation kit onto the device. After loading, identify regions of interest within the sample by staining, morphology, and location.Use immunofluorescence to outline at least 500, 000 micrometer square segments of interest. Then initiate the laser microdissection to excise the region using a laser power of greater than 40. Upon completion of the laser microdissection process, cap the microcentrifuge collection tubes and flick the tubes vigorously to ensure that the contents move from the caps to the bottoms of the tubes.After centrifugation, incubate the tubes in a 42 degrees Celsius water bath for 30 minutes before centrifuging the tubes again. Then transfer the supernatants to new 500 microliter tubes from minus 80 degrees Celsius storage. The identification of tubular sub-segments in the kidney is accomplished through antibody staining of unique tubular markers in addition to cell morphology and structural landmarks.Fluorescence labeling in conjunction with morphological tissue features and the spatial positioning of the image structures facilitates the visualization of renal sub-segments with a high degree of confidence. In this representative downstream sequencing analysis, the success rate of meeting the minimum dissection area input was greater than 90%resulting in a high total mRNA quantity available for gene detection. In this analysis, 100%of the samples met the minimum detection threshold of at least 10, 000 genes.After laser microdissection, enrichment analysis of the gene expression of known markers can be compared to those from other compartments. In this analysis, one marker for each sub-segment was identified through laser microdissection regional transcriptomics that also yielded a specific immunohistochemical staining of the corresponding nephron sub-segment. It's important to note that laser microdissection relies heavily on the expertise of the user to precisely identify the structures of interest to be dissected.This technique allows the study of specific regional gene expression signatures that can be used to assess potential pathways involved in disease progression, as well as the biology of healthy tissue.

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