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 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 analysis^2,3,4,5. 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 µm 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.

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-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.
2. Cool the cryostat to -20 °C. Clean the work surfaces and install a new cutting blade.
3. Place a small slide box (cleaned with RNase surface decontamination solution) inside the cryostat chamber to store slides with freshly cut tissue.
4. 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.
5. Cut the specimen to a thickness of 12 µm 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 °C with a desiccant cartridge and inside a tightly closed plastic bag to prevent excess moisture from accumulating inside the slide box.
6. Label each slide with a specimen ID, date, and slide number.
7. Use the slides with specimens within 10 days from the initial date of cryosectioning.

2. Laser microdissection

1. Immediately before the staining, prepare the Antibody Mix (Ab-Mix) in 10% BSA in RNase-free PBS by adding the following: 4 µL of FITC-Phalloidin, 1.5 µL of DAPI, 2 µL of Tamm-Horsfall Protein (THP) antibody directly conjugated to Alexa Fluor 546, 3.3 µL of RNase Inhibitor, and 89.2 µL of 10% BSA in PBS (to reach a volume of 100 µL).
   NOTE: Alternative antibodies may be used in place of the THP antibody. For example, 2 µL 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.
2. Wash the slide in ice cold (-20 °C) 100% acetone for 1 min and move it to the humidity chamber.
3. Wash the top of the slide with RNase-free PBS for 30 s. Repeat.
4. Wash the top of the slide with 10% BSA in RNase-free PBS for 30 s. Repeat.
5. Apply the Ab-Mix for 5 min.
6. Wash the top of the slide with 10% BSA in RNase-free PBS for 30 s. Repeat.
7. Air dry the slide for 5 min and load it onto the laser microdissection cutting platform.
8. Install the collection tubes (autoclaved 0.5 mL microcentrifuge tubes) appropriate for PCR work, containing 50 µL of Extraction Buffer from the RNA isolation kit.
9. Proceed with LMD. Complete each LMD session within at most 2 hours.
   1. 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 – 1 ng of RNA, a minimum of 500,000 µm² area is required. This often necessitates the use of up to 8 x12 µm thick sections to obtain a sufficient amount of material for all sub-segments of interest.
   2. 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.
10. Obtain a bulk cross-sectional expression signature by affixing two 12 µm sections to an LMD slide and dissecting the entire sections into extraction buffer.
11. 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
12. Centrifuge the tubes at 3,000 x g for 30 s.
13. Incubate the tubes in 42 °C water bath for 30 min.
14. Centrifuge the tubes at 3,000 x g for 2 min.
15. Transfer the supernatant to a new 0.5 mL tube and store it in -80 °C.

3. RNA isolation

NOTE: For this RNA isolation protocol, we have adapted a protocol from a commercial RNA isolation kit. The manufacturer’s protocol has been modified to meet the quality control requirements set for the project.

1. Add 250 µL of Conditioned Buffer (CB) to each RNA purification column (PC) and incubate for 5 min at room temperature.
2. Centrifuge all PCs for 1 min at 16,000 x g.
3. Add 50 µL 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.
4. 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.
5. Add 100 µL of Wash Buffer 1 (WB1) into the PCs and centrifuge for 1 minute at 8,000 x g.
6. Prepare 40 µL of DNase per each sample (Add 5 µL of DNase to 35 µL of RDD buffer). Then add 40 µL of the mixture directly on the membrane of the PC and incubate for 15 min at room temperature.
7. Add 40 µL of WB1 onto the membrane of PC, and centrifuge for 15 s at 8,000 x g.
8. Add 100 µL of Wash Buffer 2 (WB2) onto the membrane of PC, and centrifuge for 1 min at 8,000 x g.
9. Add 100 µL 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.
10. Transfer the PC to a new 0.5 mL tube.
11. Add 12 µL 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 µL per sub-segment.
12. Centrifuge the samples for 1 min at 1,000 x g and then for 2 min at 16,000 x g.
13. Transfer 2 µL into a fresh tube for Bioanalyzer analysis (to prevent freeze-thaw events).
14. Store all tubes in -80 °C until ready for further processing.

4. RNA sequencing

1. Assess each sample, intended for sequencing, for quality using a commercial Bioanalyzer and a chip dedicated to measuring small quantities of RNA.
2. 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 – 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 > 75%).
3. 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.
4. Add cDNA adapters and indexes.
5. Purify the RNAseq libraries using magnetic bead technology.
6. Deplete the ribosomal cDNA using a commercial rRNA removal kit prior to RNAseq library amplification step.
7. Purify the final RNAseq library using magnetic bead technology at a 2 ng/µL cDNA library concentration.
8. 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.
9. Use a Reference RNA (25 µg) with every sequencing run to allow for control of batch effect. The initial concentration of our Reference RNA is 1 µg/µL, while the final concentration utilized in sequencing is 25 ng/µL. Run the reference RNA as a separate sample during library preparation and run with all LMD specimens each time.
10. 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.
11. Use Integrative Genomics Viewer (IGV) for alignment and edgeR/rbamtools for transcript expression measures.
12. 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.
13. 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.
14. 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 > 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 >90% for each sequencing run.

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