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 microdissection system featuring pulsed UV laser.
1. Cryosectioning
<ol>
	<li>Expose 1.2 &#181;m LMD PPS-membrane (poly(p-phenylene sulfide) slides to UV light (in a tissue culture laminar flow hood) for 30 minutes, immediately prior to cryosectioning. Store the slides at room temperature for optimal tissue adherence.</li>
	<li>Cool the cryostat to -20 &#176;C. Clean the work surfaces and install a new cutting blade.</li>
	<li>Place a small slide box (cleaned with RNase surface decontamination solution) inside the cryostat chamber to store slides with freshly cut tissue.</li>
	<li>Adhere the specimen in OCT to a tissue holder and allow it to equilibrate for a few minutes to reach the chamber temperature and strengthen the adhesion between the OCT block and the holder. Aid the process by using a heat extractor.</li>
	<li>Cut the specimen to a thickness of 12 &#181;m and affix it to the specialized LMD slide, using the slide adapter. Each slide holds one nephrectomy section per slide or up to two kidney biopsy sections per slide. Store the slides at -80 &#176;C with a desiccant cartridge and inside a tightly closed plastic bag to prevent excess moisture from accumulating inside the slide box.</li>
	<li>Label each slide with a specimen ID, date, and slide number.</li>
	<li>Use the slides with specimens within 10 days from the initial date of cryosectioning.</li>
</ol>
2. Laser microdissection
<ol>
	<li>Immediately before the staining, prepare the Antibody Mix (Ab-Mix) in 10% BSA in RNase-free PBS by adding the following: 4 &#181;L of FITC-Phalloidin, 1.5 &#181;L of DAPI, 2 &#181;L of Tamm-Horsfall Protein (THP) antibody directly conjugated to Alexa Fluor 546, 3.3 &#181;L of RNase Inhibitor, and 89.2 &#181;L of 10% BSA in PBS (to reach a volume of 100 &#181;L). 
	NOTE: Alternative antibodies may be used in place of the THP antibody. For example, 2 &#181;L of megalin/LRP2 antibody, directly conjugated to Alexa Fluor 568, can be used to label the proximal tubule. The Ab-Mix contains either LRP2 or THP antibody (to visualize either proximal tubules or thick ascending limbs, respectively). Other antibodies may be validated according to user needs.</li>
	<li>Wash the slide in ice cold (-20 &#176;C) 100% acetone for 1 min and move it to the humidity chamber.</li>
	<li>Wash the top of the slide with RNase-free PBS for 30 s. Repeat.</li>
	<li>Wash the top of the slide with 10% BSA in RNase-free PBS for 30 s. Repeat.</li>
	<li>Apply the Ab-Mix for 5 min.</li>
	<li>Wash the top of the slide with 10% BSA in RNase-free PBS for 30 s. Repeat.</li>
	<li>Air dry the slide for 5 min and load it onto the laser microdissection cutting platform.</li>
	<li>Install the collection tubes (autoclaved 0.5 mL microcentrifuge tubes) appropriate for PCR work, containing 50 &#181;L of Extraction Buffer from the RNA isolation kit.</li>
	<li>Proceed with LMD. Complete each LMD session within at most 2 hours.
	<ol>
		<li>Collect pre- and post-LMD immunofluorescence images, using the microscope camera to validate the dissection for inter-operator variability as well as archival purposes, training and quality assessment of the performed protocol. In order to obtain 0.5 &#8211; 1 ng of RNA, a minimum of 500,000 &#181;m2 area is required. This often necessitates the use of up to 8 x12 &#181;m thick sections to obtain a sufficient amount of material for all sub-segments of interest.</li>
		<li>Identify regions of interest by staining, morphology and location and excise them using laser power greater than 40. 
		NOTE: Here are the dissection criteria. The proximal tubule is defined by FITC-Phalloidin and LRP2 labeling. The thick ascending limb is defined by THP labeling. The collecting duct is defined by nuclear morphology (DAPI) and absence of other staining. The glomerulus is defined by FITC-Phalloidin and morphology. The interstitium is defined as the area between stained tubules.</li>
	</ol>
	</li>
	<li>Obtain a bulk cross-sectional expression signature by affixing two 12 &#181;m sections to an LMD slide and dissecting the entire sections into extraction buffer.</li>
	<li>Upon completion of the LMD process, close the collecting microcentrifuge tubes and flick it vigorously to ensure that the content moved from the cap to the bottom of the tube</li>
	<li>Centrifuge the tubes at 3,000 x g for 30 s.</li>
	<li>Incubate the tubes in 42 &#176;C water bath for 30 min.</li>
	<li>Centrifuge the tubes at 3,000 x g for 2 min.</li>
	<li>Transfer the supernatant to a new 0.5 mL tube and store it in -80 &#176;C.</li>
</ol>
3. RNA isolation
NOTE: For this RNA isolation protocol, we have adapted a protocol from a commercial RNA isolation kit. The manufacturer&#8217;s protocol has been modified to meet the quality control requirements set for the project.
<ol>
	<li>Add 250 &#181;L of Conditioned Buffer (CB) to each RNA purification column (PC) and incubate for 5 min at room temperature.</li>
	<li>Centrifuge all PCs for 1 min at 16,000 x g.</li>
	<li>Add 50 &#181;L of 70% ethanol (provided in the Kit) into the tubes with tissue samples. Mix the samples well by pipetting up and down. Do not vortex. Do not centrifuge.</li>
	<li>Transfer the mixture into conditioned PCs and centrifuge for 2 min at 100 x g (to bind RNA), quickly follow with centrifugation for 30 s at 16,000 x g (to remove flow through). Repeat this step if more than 1 tube with tissue samples are available for any given sub-segment.</li>
	<li>Add 100 &#181;L of Wash Buffer 1 (WB1) into the PCs and centrifuge for 1 minute at 8,000 x g.</li>
	<li>Prepare 40 &#181;L of DNase per each sample (Add 5 &#181;L of DNase to 35 &#181;L of RDD buffer). Then add 40 &#181;L of the mixture directly on the membrane of the PC and incubate for 15 min at room temperature.</li>
	<li>Add 40 &#181;L of WB1 onto the membrane of PC, and centrifuge for 15 s at 8,000 x g.</li>
	<li>Add 100 &#181;L of Wash Buffer 2 (WB2) onto the membrane of PC, and centrifuge for 1 min at 8,000 x g.</li>
	<li>Add 100 &#181;L of WB2 onto the membrane of PC, centrifuge for 2 min at 16,000 x g, immediately follow by centrifugation for 1 min at 16,000 x g.</li>
	<li>Transfer the PC to a new 0.5 mL tube.</li>
	<li>Add 12 &#181;L of Elution Buffer (EB) onto the membrane and incubate for 7 min at room temperature. Thus, the final volume of all pooled dissected tissue samples is 12 &#181;L per sub-segment.</li>
	<li>Centrifuge the samples for 1 min at 1,000 x g and then for 2 min at 16,000 x g.</li>
	<li>Transfer 2 &#181;L into a fresh tube for Bioanalyzer analysis (to prevent freeze-thaw events).</li>
	<li>Store all tubes in -80 &#176;C until ready for further processing.</li>
</ol>
4. RNA sequencing
<ol>
	<li>Assess each sample, intended for sequencing, for quality using a commercial Bioanalyzer and a chip dedicated to measuring small quantities of RNA.</li>
	<li>Following quality control (QC) parameters prior to library prep and sequencing are required: Quantity of RNA greater than 4 ng for bulk and greater than 0.5 &#8211; 1 ng for each sub-segment; The percent of transcripts longer than 200 nucleotides (DV200) is required to be greater than 25% for LMD specimens (optimal &#62; 75%).</li>
	<li>Carry out library prep with a commercial cDNA library preparation kit intended for small quantities of degraded RNA. For the commercial kit listed in the supplement, we suggest using Option 2, which requires a minimum DV200 of 25% and no fragmentation. Some sequencing technologies may require higher minimum DV200 thresholds, such as 30%7.</li>
	<li>Add cDNA adapters and indexes.</li>
	<li>Purify the RNAseq libraries using magnetic bead technology.</li>
	<li>Deplete the ribosomal cDNA using a commercial rRNA removal kit prior to RNAseq library amplification step.</li>
	<li>Purify the final RNAseq library using magnetic bead technology at a 2 ng/&#181;L cDNA library concentration.</li>
	<li>Carry out RNA sequencing of 75 bp paired end on a commercial sequencing system with 30 million reads/sample for bulk and 100 million reads/sample for sub-segmental sections.</li>
	<li>Use a Reference RNA (25 &#181;g) with every sequencing run to allow for control of batch effect. The initial concentration of our Reference RNA is 1 &#181;g/&#181;L, while the final concentration utilized in sequencing is 25 ng/&#181;L. Run the reference RNA as a separate sample during library preparation and run with all LMD specimens each time.</li>
	<li>Perform data analysis utilizing the FastQC application to assess the quality of sequencing, intergenic and mitochondrial reads and to determine reads attributed to a gene.</li>
	<li>Use Integrative Genomics Viewer (IGV) for alignment and edgeR/rbamtools for transcript expression measures.</li>
	<li>Remove samples with less than 100,000 reads. Quantile normalize the raw reads in the data set after filtering out lowly expressed genes at a user defined threshold.</li>
	<li>Quantify the expression as a ratio of the sub-segment of interest to the average of all other sub-segments and log2-transform. Relative expression of the same gene may be compared across sub-segments and samples; however, it is not ideal to compare relative expression between two different genes due to potential differences in degradation across genes and RNA species.</li>
	<li>Carry out an enrichment analysis to compare gene expression for a set of makers specific to each nephron sub-segment, while Reference RNA samples are compared across batches. A batch effect within 1 standard deviation of mean expression with R value &#62; 0.9 is considered acceptable. Additional normalization is required for a higher batch effect score. Any run that deviates from the accepted batch effect can be flagged. The Q30 should be greater than &#62;90% for each sequencing run.</li>
</ol>

<ol>
	<li>El-Serag, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+in+Barrett's+esophagus:+laser+capture+versus+whole+tissue.">Gene expression in Barrett's esophagus: laser capture versus whole tissue.</a> Scandinavian Journal of Gastroenterology. 44 (7), 787-795 (2009).</li><li>Kohda, Y., Murakami, H., Moe, O. W., Star, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+segmental+renal+gene+expression+by+laser+capture+microdissection.">Analysis of segmental renal gene expression by laser capture microdissection.</a> Kidney International. 57 (1), 321-331 (2000).</li><li>Murakami, H., Liotta, L., Star, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IF-LCM:+laser+capture+microdissection+of+immunofluorescently+defined+cells+for+mRNA+analysis+rapid+communication.">IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication.</a> Kidney International. 58 (3), 1346-1353 (2000).</li><li>Woroniecki, R. P., Bottinger, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+kidney+tissue.">Laser capture microdissection of kidney tissue.</a> Methods in Molecular Biology. 466, 73-82 (2009).</li><li>Noppert, S. J., Eder, S., Rudnicki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-capture+microdissection+of+renal+tubule+cells+and+linear+amplification+of+RNA+for+microarray+profiling+and+real-time+PCR.">Laser-capture microdissection of renal tubule cells and linear amplification of RNA for microarray profiling and real-time PCR.</a> Methods in Molecular Biology. 755, 257-266 (2011).</li><li>Amini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimised+protocol+for+isolation+of+RNA+from+small+sections+of+laser-capture+microdissected+FFPE+tissue+amenable+for+next-generation+sequencing.">An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.</a> BMC Molecular Biology. 18 (1), 22 (2017).</li><li>. Catalytic FFPE Nucleic Acid Isolation for best NGS Performance Available from: <a target="_blank" href="https://celldatasci.com/products/RNAstorm/RNAstorm_Technical_Note.pdf">https://celldatasci.com/products/RNAstorm/RNAstorm_Technical_Note.pdf</a> (2016)</li><li>Micanovic, R., Khan, S., El-Achkar, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+laser+micro-dissection+of+specific+nephron+segments+in+the+mouse+kidney+allows+targeted+downstream+proteomic+analysis.">Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis.</a> Physiological Reports. 3 (2), (2015).</li><li>Lake, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-nucleus+RNA-sequencing+pipeline+to+decipher+the+molecular+anatomy+and+pathophysiology+of+human+kidneys.">A single-nucleus RNA-sequencing pipeline to decipher the molecular anatomy and pathophysiology of human kidneys.</a> Nature Communications. 10 (1), 2832 (2019).</li><li>Rodriguez-Canales, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+molecular+profiling+of+tissue+and+tissue+components:+defining+the+best+processing+and+microdissection+methods+for+biomedical+applications.">Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.</a> Methods in Molecular Biology. 980, 61-120 (2013).</li><li>Hipp, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-Aided+Laser+Dissection:+A+Microdissection+Workflow+Leveraging+Image+Analysis+Tools.">Computer-Aided Laser Dissection: A Microdissection Workflow Leveraging Image Analysis Tools.</a> Journal of Pathology Informatics. 9, 45 (2018).</li></ol>

The authors have nothing to disclose.

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 ...

application-laser-microdissection-to-uncover-regional-transcriptomics

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3.8K Views.  Indiana University School of Medicine. 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.

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

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Generation of Human Kidney Tubuloids from Tissue and Urine

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high efficiency. Normal kidney tubuloids recapitulate many aspects of their tissue of origin. They represent distinct nephron segments - most notably of the proximal tubule, loop of Henle, distal tubules, and collecting duct - and can be used to study normal kidney physiology. Furthermore, tubuloid technology facilitates disease modeling, e.g., for infectious diseases as well as for cancer. Obtaining kidney epithelial cells for tubuloid generation is, however, dependent on leftover surgical material (such as partial) nephrectomies) or needle biopsies. The ability to grow tubuloids from urine would provide an alternative, less invasive source of healthy kidney epithelial cells. It has been previously shown that tubuloid cultures can be successfully generated from only a few milliliters of freshly collected urine. This article describes the protocols to generate and propagate ASC-derived human kidney tubuloid cultures from tissue and urine samples.

Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...