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.
