This work was funded by the PhD studentship for Joseph Morgan from Alzheimer&#39;s Society UK (Grant number 549/SERA-52) and by the Innovation Strategy funds of the University of Salford (grant SEFA-39). The brain tissue was obtained from the Manchester brain bank (REC Reference 09/H0906/52) of the Brains for Dementia Network.

The work has been ethically approved by the Manchester Brain Bank (REC reference 09/H0906/52) and by the ethics committee at the University of Salford (Application ID: 3408).
1. Breakdown of intracellular matrix using collagenase on frozen brain tissue sections
<ol>
	<li>Thinly slice 250 mg frozen brain tissue(around 0.4 mm thick fragments) using a sterilized scalpel on a cold plate and add 2 mL of 75 U/mL collagenase type-III/ Hibernate-E solution.</li>
	<li>Incubate each sample in a water bath at 37 &#176;C for 20 min. At the 5 min point, invert each sample two times to mix; at the 10 min point, pipette each sample three times gently using a 10 mL plastic disposable stripette.</li>
	<li>Place each sample on ice and add 1x Protease inhibitor cocktail and 1x phosphatase inhibitor. This stops the enzyme reaction and is the endpoint of digestion using collagenase type-III.</li>
	<li>Centrifuge each brain tissue sample at 300x g for 10 min at 4 &#176;C. Collect each supernatant and further centrifuge at 2000 x g for 15 min at 4 &#176;C.</li>
	<li>Collect each supernatant again and filter through a 0.22 &#181;m filter. Centrifuge each filtrate at 10 000 x g for 48 min at 4 &#176;C.</li>
	<li>Collect the supernatant and add precipitation buffer at a ratio of 2:1 to each sample. Incubate overnight at 4 &#176;C.</li>
	<li>Centrifuge each sample at 10 000 x g for 96 min at 4 &#176;C and resuspend the pellet in 100 &#181;L of extracellular vesicle (EV) free PBS.</li>
</ol>
2. Preparation of size exclusion chromatography columns
<ol>
	<li>Prior to use, equilibrate the pre-prepared SEC column at room temperature (RT) for 15 min. 
	NOTE: The bottom cap must be removed before the screw cap.</li>
	<li>After equilibration, remove the preservative buffer from the top of the column and wash the column twice using 250 &#181;L of EV-free PBS. Each PBS wash is left to enter the column through gravity.</li>
</ol>
3. Isolation of small extracellular vesicles using size exclusion chromatography
<ol>
	<li>Add the resuspended pellet to the top of the SEC column and centrifuge the column at 50 x g for 30 s. Discard the flow-through.</li>
	<li>Add 180 &#181;L of EV-free PBS to the top of the SEC column and centrifuge the column at 50 x g for 1 min, allowing the brain-derived sEVs to elute from the column. 
	NOTE: The part of the protocol detailed above is summarized in Figure 1.</li>
</ol>
4. Confirmation of small extracellular vesicle markers by western blotting
<ol>
	<li>To lyse sEVs prior to western blot analysis (to ensure all membrane and cytosolic cargoes are analyzed), dilute isolated sEV sample 1:1 in lysis and extraction buffer (1x protease inhibitor and 1x phosphatase inhibitor).</li>
	<li>Agitate the sample at 4 &#176;C for 30 min.</li>
	<li>Centrifuge sample at 14 000 x g for 15 min.</li>
	<li>Collect the protein supernatant and measure protein concentrations using a protein assay kit.</li>
	<li>Mix samples with 4x Laemmli buffer and load 20 &#181;g of protein into each well against a pre-stained protein ladder.</li>
	<li>Once resolved, transfer the proteins onto a 0.45 &#181;m nitrocellulose membrane. After the transfer, block the membrane for 2 h in 5% BSA.</li>
	<li>Incubate the membranes with primary antibodies (Table 1) overnight.</li>
	<li>Wash the membrane three times with wash buffer (1% Tween20 in PBS) and stain using an anti-mouse secondary antibody. Afterward, wash three times with wash buffer (1% Tween20 in PBS).</li>
	<li>Follow the above steps in the manuscript before imaging the blots.</li>
	<li>Image using horseradish peroxidase (HRP) Substrate.</li>
</ol>
5. Confirmation of small extracellular vesicles by nanoparticle tracking analysis (NTA)
<ol>
	<li>Perform NTA to analyze the concentration and size of all particles in the sample. Carry out this by diluting each sample in PBS at a dilution factor of 1:1000.</li>
	<li>Inject the sample into the NTA instrument and read the sample at a wavelength of 550 nm. At this stage, use the particle quantity to measure the amount of protein per particle (usually expressed in femtogram) as recommended in MISEV guidelines.</li>
	<li>Next, for fluorescent NTA, dilute cell mask orange (CMO) (stains all biological particles within a sample) in PBS by a dilution factor of 1:1000. From this, dilute CMO dye using sEV sample by a dilution factor of 1:10 - Incubate this dilution step in the dark for 30 min at 4 &#176;C.</li>
	<li>Dilute the second CMO dilution using PBS by a dilution factor of 1:1000. 
	NOTE: The final dilution of CMO dye must be 1:10 000 000.</li>
	<li>Read the CMO of each sEV sample at a wavelength of 550 nm using the NTA instrument.</li>
	<li>For the tetraspanin fluorescent antibodies (see Table of Materials), firstly, dilute each respective antibody in PBS by a dilution factor of 1:10. From this, dilute each of the dyes using sEV sample by a dilution factor of 1:10 - Incubate the dilution step in the dark for 2 h at 4 &#176;C.</li>
	<li>Dilute the second antibody dilution using PBS by a dilution factor of 1:1000. 
	NOTE: The final dilution of each tetraspanin antibody must be 1:100 000.</li>
	<li>Read the fluorescent antibody readings of the sEV sample at a wavelength of 550 nm using the NTA instrument.</li>
</ol>
6. Confirmation of small extracellular vesicles by transmission electron microscopy (TEM)
NOTE: This protocol was performed by the Biomedical Microscopy Facility at the University of Liverpool.
<ol>
	<li>Firstly, fix sEV samples using glutaraldehyde and stain using uranyl acetate.</li>
	<li>Through a graded series of alcohols, dehydrate the sample ready for TEM.</li>
	<li>Image samples using a suitable Transmission Electron Microscope.</li>
</ol>
7. Proteinase K and RNase A treatment
<ol>
	<li>To 50 &#181;L of isolated brain derivedsEVs, add 1 &#181;L of 20 &#181;g/&#181;L proteinase K and incubate at 37 &#176;C for 30 min.</li>
	<li>Stop each reaction by adding 1x proteinase inhibitor cocktail and incubate on ice for 10 min.</li>
	<li>After incubation, add 1 &#181;L of 10 &#181;g/&#181;L RNase A to each sample and further incubate at 37 &#176;C for 30 min. 
	NOTE: Immediately move on to section 8.</li>
</ol>
8. Total RNA isolation from small extracellular vesicles
<ol>
	<li>To isolate total RNA, add 700 &#181;L of high-quality RNA recovery lysis reagent to 50 &#181;L of brain-derived EV (BDsEV) sample. Agitate each sample for 1 h.</li>
	<li>Add 140 &#181;L of chloroform, shake each sample vigorously for 20 s, and leave to incubate at RT for 3 min.</li>
	<li>Centrifuge the sample at 12 000 x g for 15 min at 4 &#176;C and collect the interphase from the sample. Add 1.5x volume of 100% ethanol.</li>
	<li>Add 700 &#181;L of interphase/ethanol mixture RNA isolation columns and centrifuge at &#8805;8000 x g for 15 s at RT. 
	NOTE: Repeat this step using all the interphase/ethanol samples.</li>
	<li>To the column, add 700 &#181;L of wash buffer and centrifuge at &#8805;8000 x g for 15 s. Discard the flow-through.</li>
	<li>Add 500 &#181;L of buffer RPE and again centrifuge at &#8805;8000 x g for 15 s. Discard the flow-through.</li>
	<li>Add 500 &#181;L of 80% ethanol and centrifuge at &#8805;8000 x g for 2 min. Discard the flow-through.</li>
	<li>At this point, dry the column membrane by centrifuging the columns at full speed (&#62;8000 x g) for 5 min with the lid open.</li>
	<li>Transfer the column to a new collection tube, add 14 &#181;L of RNase-free water directly to the column membrane, and centrifuge at &#8805;8000 x g for 1 min to elute total RNA from BDsEVs.</li>
	<li>Quantify the total microRNA with samples using a miRNA quantification assay kit, where samples were measured on a quantification fluorometer, selecting the miRNA assay type.</li>
</ol>
9. Small RNA sequencing
<ol>
	<li>After quantification of miRNA within the sample, synthesize a cDNA library using the Small RNA-Seq Library Prep Kit. 
	NOTE: Using this protocol, library generation included four steps: 3&#39; adapter ligation, purification, 5&#39; adapter ligation, and reverse transcription - where, in the end, an RNA/cDNA library is formed. After library generation, endpoint PCR amplification occurs with index primers being added (used for multiplexing purposes), after which the samples are purified.</li>
	<li>To test for RNA quality, use a quality control application assay detailing the size of each preparation and the peak intensity.</li>
	<li>To test for DNA concentration, measure the DNA quantification on a quantification fluorometer, selecting the dsDNA assay type.</li>
	<li>Prepare the samples for Small RNA Seq using the reagent kit, after which the samples are loaded into the flow cell.</li>
</ol>

Efficient Isolation of Brain sEVs from Minimal Tissue for Biomarker Discovery

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There are no conflicts of interest for any of the authors.

This modified and improved protocol for&#160;isolating brain-derived small extracellular vesicles and their microRNA cargo&#160;demonstrates the feasibility of using minimal&#160;tissue without compromising the quality and&#160;quantity of the products downstream.&#160;In the field of biomarker discovery, identifying molecular identifiers that are specific to cell and tissue types can lead to more accessible means of non-invasive diagnostic tests using body fluids. Furthermore, the approach described here provides the framework for the identification of specific sEV subpopulations within different cell types (neuronal vs. glial), tissue types (hippocampal tissue vs prefrontal cortex tissue), and biofluids - which will offer a greater understanding of cargo loading signatures, pathophysiology and disease extent. Previously outlined in Vella et al., it was suggested that 450 mg to 1 g should be used for BDsEV isolation, coupled with density gradient ultracentrifugation (using speeds of up to 180,000 x g)8. In this methodology, sEVs have successfully been isolated using lower amounts (&#62;50% reduction) of fresh-frozen brain tissue samples (250-450 mg). The reduction of the amount of brain tissue used to isolate sEVs leads to reduced costs and consumables. Equally, the protocol has been made more accessible through the application of SEC coupled with high-speed centrifugation (10,000 x g) instead of ultracentrifugation (180,000 x g).

Using fNTA and TEM, we established that the size range profile of the sEVs falls within the characteristic 30-200 nm size range, as recommended by the MISEV guidelines13,14, with TEM providing morphological confirmation of the sEVs obtained. As shown in Figure 3C and in Supplementary File 4, &#62;87% of the particles are &#60;250 nm size range, and the samples, on average, show 10.56 fg protein/BDsEV. Specific species of small extracellular vesicles vary in size and shape, where characteristically smaller subpopulations, such as exosomes, are observed to have a uniform morphology in comparison to microvesicles that are said to have an irregular structure due to their nature of biogenesis15. For the fNTA, a scatter reading (520 nm) was produced to analyze the total particles in samples, providing details of size profile and concentrations. The readings show that over 50% of the particles were biological (through the staining of CMO), with populations of biological particles that fall within the size range of sEVs displaying tetraspanin markers, with CD9 being the most predominant in our control samples (34.66%). Whereas for western blotting, the presence&#160;of characteristic membrane and cytosolic&#160;protein cargoes provided evidence of sEV&#160;presence, with the absence of Calnexin&#160;confirming BDsEVs to be free of any cellular&#160;contamination.&#160;To further ensure that the miRNA available for downstream processing was part of sEV cargo (and not other extracellular RNA), we treated the sEVs with Proteinase K and RNase A enzymes before disrupting the sEV membranes to degrade any protein and RNA that were externally present in the extracellular space outside the BDsEVs16.
Total RNA was extracted from the sEVs and prepared for downstream applications such as qRT-PCR and next-generation sequencing (for small RNA). Standard checks using quality control assays showed high-quality samples and cDNA libraries ready for sequencing. The cDNA libraries peak at around 120-160 bp, attributing to miRNA-derived cDNA peaks with adaptor sequences of varying lengths, with any smaller peaks potentially representing adaptor dimers.
Analysis of the next-generation sequencing data using FASTQC and CutAdapt shows high-quality results obtained from BDsEV miRNAs. As shown in Figure 5, each position read has a Q score higher than 30 (Q30), showing an error probability of 0.001-0.0001 (1 in 1000-10,000). Moreover, sequence length distribution analysis (Figure 6) shows predominant peaks falling within the characteristic range of miRNA (19-25 bp), suggesting high quality and specificity for the data obtained.
Numerous future studies will benefit from this method due to its improved accessibility, alongside saving cost and resources through reducing not only the amount of tissue samples, but also saving on the cost of reagents. The reduction (&#62;50%) in the required amount of tissue makes this method also more ethical. Aligned with the global efforts in precision health, the improved workflow detailed here provides comparatively more affordable tools to embark on accurate cell type-specific biomarkers via non-invasive (or minimally invasive) routes using BDsEVs and their cargo. This can be used for a broad range of neurological conditions. A possible limitation of this method is that the protocol will need adjustment if researchers want to use a tissue other than the human brain for sEV isolation. In the long run, this improved method will make non-invasive, tissue-specific biomarker discovery and validation more equitable and accessible around the world.

Extracellular vesicles (EVs) are one of the key players of inter-cellular communication in all multicellular organisms1. EVs are cell-derived lipid bilayer membrane particles that can facilitate the transfer of a variety of cargo loads, such as proteins, lipids, and nucleic acids, to recipient cells. EVs can have a broad size range ranging from 30 nm up to 1 &#181;M. Small EVs (sEVs), defined as lipid-bound vesicles with an average diameter size of &#60;200 nm, have the ability to cross the blood-brain barrier; therefore, they have been implicated in the prion-like spread and exacerbation of neurodegenerative diseases and other conditions such as Alzheimer&#39;s disease (AD), frontotemporal dementia (FTD), Parkinson&#39;s disease (PD) and cancers2,3. Furthermore, as sEVs can be found in a range of biofluids such as blood, cerebrospinal fluid (CSF), saliva, and even urine, their beneficial use can span the field of biomarkers and non-invasive diagnostics. For example, AD research may point to the use of biofluid pathogenic protein ratios, such as tau/A&#946;, or even A&#946;42/A&#946;404.
One known cargo of sEVs is microRNA (miRNA), a group of small, non-coding RNA molecules of around 22 nucleotides in length that bind to the 3&#39;-UTR regions of mRNA and usually negatively regulate protein expression. Involved in many cellular roles, miRNAs have also been implicated in the pathogenesis of various diseases, including cancers and neurodegenerative diseases. Cheng et al. conducted a high-throughput sequencing analysis of serum-derived sEV miRNA expression signatures from AD patients5. Once coupled with neuroimaging records and known risk factors of age, sex, and APOE &#949;4 allele presentation, results were found to predict AD with 87% sensitivity and 77% specificity. Moreover, research have identified two upregulated CSF miRNAs (miR-151a-3p, let-7f-5p) and 3 downregulated miRNAs (miR-27a-3p, miR-125a-5p and miR-423-5p) that can potentially diagnose early-stage PD6. With pathological diseases, the pathological status may precede certain characteristic symptoms of diseases, whereas in neurodegeneration, the accumulation of pathological hallmarks occurs much earlier than cognitive decline.
MicroRNAs are potentially a more effective biomarker than proteins, owing to their diverse functions, and higher-order epigenetic regulation. Using brain tissue, researchers can potentially identify specific brain-derived sEV (BDsEV) miRNA signatures for diseases and their subtypes. For example, sEVs with neuronal and glial markers can present different miRNA cargo, and the analysis can result in more precise methods of disease detection. Furthermore, BDsEVs are suggested to play a large role in the transsynaptic spread of neuropathogenic proteins7. Previous reports have suggested immunoprecipitation and density gradient (sucrose gradient) ultracentrifugation to obtain sEVs from fresh-frozen brain tissue8,9. However, these approaches require specific infrastructure with ultracentrifuge and downstream purification methods to obtain high-quality sEV samples10. More recent reports have suggested several modifications and improvements to the approach11,12,13; however, despite this, isolation and study of sEV-derived microRNA from human tissues are still not widely applied. The approach described in this protocol aims to provide a refined, step-by-step protocol for brain-derived sEV study to enhance accessibility to this technique. We established a protocol using minimal&#160;amounts of brain tissue, from which we&#160;isolated pure sEVs using size exclusion chromatography and show high-quality next-generation sequencing data from the microRNA&#160;cargo of these sEVs.

<table><tbody><tr><td>Bovine Serum Albumin</td><td>Merck</td><td>A9418-100G</td><td></td></tr><tr><td>Cell Mask Orange Plasma Membrane Stain</td><td>ThermoFisher Scientific</td><td>C10045</td><td></td></tr><tr><td>Collagenase Type-III</td><td>StemCell Technologies</td><td>07422</td><td></td></tr><tr><td>ExoSpin Columns and Buffer</td><td>Cell Guidance Systems Ltd.</td><td>EX01-50</td><td>This kit contains SEC columns used in this experiment, precipitation buffer and EV free PBS.</td></tr><tr><td>Halt Protease Inhibitor Cocktail (100x)</td><td>ThermoFisher Scientific</td><td>78429</td><td></td></tr><tr><td>Hibernate-E Medium</td><td>ThermoFisher Scientific</td><td>A1247601</td><td></td></tr><tr><td>Laemmli Sample Buffer (4x)</td><td>BioRad</td><td>1610747</td><td></td></tr><tr><td>Lexogen Small RNA-Seq Library Prep Kit</td><td>Lexogen</td><td>052.24</td><td>This kit contains Small RNA preparation reagent box with i7 Index primer plate.&amp;nbsp;</td></tr><tr><td>miRNeasy Micro Kit (50)</td><td>Qiagen</td><td>217084</td><td>This kit contains high-quality RNA recovery lysis reagent (Qiazol), RNA isolation columns, isolation buffers (RWT, RPE) and RNase free water.</td></tr><tr><td>MiSeq Reagent Kit v3</td><td>Illumina</td><td>MS-102-3001</td><td>This is the Illumina Preparation Kit</td></tr><tr><td>Nitrocellulose Membrane, 0.45 &amp;mu;m</td><td>ThermoFisher Scientific</td><td>88018</td><td></td></tr><tr><td>PE/Dazzle 594 anti-human CD63 Antibody</td><td>BioLegend</td><td>143914</td><td>Used for fNTA Analysis</td></tr><tr><td>PE/Dazzle 594 anti-human CD81 Antibody</td><td>BioLegend</td><td>349520</td><td>Used for fNTA Analysis</td></tr><tr><td>PE/Dazzle 594 anti-human CD9 Antibody</td><td>BioLegend</td><td>312118</td><td>Used for fNTA Analysis</td></tr><tr><td>PhosSTOP</td><td>Merck</td><td>4906845001</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23225</td><td></td></tr><tr><td>Proteinase K</td><td>ThermoFisher Scientific</td><td>25530049</td><td></td></tr><tr><td>Qubit microRNA Assay Kit</td><td>ThermoFisher Scientific</td><td>Q32880</td><td></td></tr><tr><td>Qubit 1X dsDNA HS assay kit</td><td>ThermoFisher Scientific</td><td>Q33230</td><td></td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>ThermoFisher Scientific</td><td>Q33216</td><td></td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>ThermoFisher Scientific</td><td>89901</td><td></td></tr><tr><td>RNase A</td><td>ThermoFisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>SuperSignal West Femto Maximum Sensitivity Substrate</td><td>ThermoFisher Scientific</td><td>34094</td><td></td></tr></tbody></table>

harnessing the power of microrna cargoes in small extracellular vesicles released from fresh-frozen human brain sections

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic acids (microRNA, mRNA, DNA). The microRNA sEV cargo has potential utility as a powerful non-invasive disease biomarker due to sEV&#39;s ability to traverse biological barriers (e.g., blood-brain barrier) and become accessible through various body fluids. Despite numerous studies on sEV biomarkers in body fluids, identifying tissue or cell-specific sEV subpopulations remains challenging, particularly from the brain. Our study addresses this challenge by adapting existing methods to isolate sEVs from minimal amounts of frozen human brain sections using size exclusion chromatography (SEC).
After ethical approval, approximately 250 &#181;g of fresh-frozen human brain tissue (obtained from Manchester Brain Bank [UK]) was sliced from the 3 donor tissues and incubated in collagenase type 3/Hibernate-E solution, with intermediate agitation, followed by serial centrifugation and filtration steps. Then, sEVs were isolated using the SEC method and characterized by following MISEV guidelines. Before isolating RNA from within these sEVs, the solution was treated with Proteinase-K and RNase-A to remove any non-sEV extracellular RNA. The RNA quantity and quality were checked and processed further for qPCR and small RNA sequencing experiments.
The presence of sEVs was confirmed through fluorescence nanoparticle tracking analysis (fNTA) and western blot for surface markers (CD9, CD63, CD81). Size distribution (50-200 nm) was confirmed by NTA and electron microscopy. The total RNA concentration within lysed sEVs ranged from 3-9 ng/&#181;L and was used for successful quantification by qPCR for selected candidate microRNAs. Small RNA sequencing on MiSeq provided high-quality data (Q &#62;32) with 1.4-5 million reads per sample.
This method enables efficient isolation and characterization of sEVs from minimal brain tissue volumes, facilitating non-invasive biomarker research and holds promise for equitable disease biomarker studies, offering insights into neurodegenerative diseases and potentially other disorders.

To confirm the presence of BDsEVs, three techniques were utilized: western blotting, NTA, and TEM (Figure 3). Western blot results (Figure 3A and Supplementary File 1) show the presence of all five positive markers (CD9, CD63, CD81, Flot-1, and TSG101) and the absence of Calnexin in sEVs (used as a negative control), confirming no contamination with cellular contents. As expected, the brain homogenates (BH) show more protein than observed in the sEVs for the markers tested. TEM images confirmed the morphology of BDsEVs and their relative size within the sample (Figure 3B), which was further confirmed by nanoparticle tracking analysis (Figure 3C).
Figure 3C (upper panel) shows the isolated particle sizes falling between 50-200 nm. From the NTA data, taking the scatter result as 100% of the represented particles, CMO represents around 52.35% of particles (Figure 3C, lower panel) to have a lipid bilayer. Of these CMO-stained particles, 34.66% contained CD9, 15.49% contained CD63, and 12.01% contained CD81. Refer to Supplementary File 2 for NTA control data.
Quantification data of isolated RNA (Table 2) show levels ranging from 3.24-8.76 (ng/&#181;L), values which were normalized when generating the cDNA libraries. TapeStation assay results (Figure 4) show major peaks of around 120-160 bp, with smaller peaks below this of around 50 bp and other smaller peaks that are higher of around 484 bp.
Quality checks for the next-generation sequencing data show very high-quality reads after adapter trimming (Figure 5A). All sequence positions show a Phred score of &#62;30, indicating error rates of less than 1 in 1000 nucleotides. Figure 5B shows the most sequences to be around 21-22 bp, matching the expected size range for microRNAs. Through further downstream mapping (using BowTie2), using the Samtools flagstat analysis biotool, out of 1,304,100 aligned sequences, 1,116 264 (85%) were mapped to the microRNA regions in the human genome (build hg38; Table 3).
Of the total miRNAs, 808 miRNAs were expressed through all 3 samples, with 344 miRNAs shared between all three samples (~43%), 5 shared between control 1 and control 2, 183 shared between control 1 and control 3, and 16 shared between control 2 and control 3 (Figure 6). From this data, the highest expressed miRNA across all three BDsEVs are hsa-let-7b-5p, hsa-miR-143-3p, hsa-miR-30a-5p, hsa-miR-221-3p and hsa-let7i-5p. Equally, the top 5 least differentially expressed miRNA across BDsEVs samples (reads &#62;10) are hsa-miR-128-2-5p, hsa-miR-182-5p, hsa-miR-193b-5p, hsa-miR-448 and hsa-miR-505-3p. All raw and normalized read counts are given in Supplementary File 3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67379/67379fig01.jpg" /> 
Figure 1: Tissue processing workflow. The figure shows the stepwise process for the isolation of brain-derived sEVs (BDsEVs) from fresh-frozen brain sections using size exclusion chromatography. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/67379fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67379/67379fig02.jpg" /> 
Figure 2: RNA isolation workflow from BDsEVs. The figure shows the stepwise process of isolating total RNA from the BDsEVs, including the pre-treatment step of removing any extracellular RNA using proteinase K and RNase A. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/67379fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67379/67379fig03.jpg" /> 
Figure 3: BDsEV characterisation workflow. The figure shows various methods to characterize the BDsEVs. (A) Western blot to detect sEV-specific protein markers. BH Brain homogenate. Calnexin is a negative control for sEVs - absence in sEV and presence in BH confirms the sEV sample purity. For other markers, as expected, the protein quantity is higher in BH than in the sEV samples (20 &#181;g of protein loaded per well). (B) Representative transmission electron microscopy (TEM) image for BDsEVs at 200 &#181;M scale, confirming the expected morphology with a lipid bilayer membrane. (C) Size distribution plot for Nanoparticle Tracking Analysis (NTA) for all particles (upper panel). Each data point represents the variation across three replicates. Normalized proportions for biological particles (sEVs) and relative proportions for specific surface markers (lower panel) (data shown as mean &#177; SEM of triplicate wells (n = 3)). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/67379fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67379/67379fig04.jpg" /> 
Figure 4: Quality check for cDNA library preparation. Tapestation analysis for individual samples after cDNA library preparation. The peaks displayed in each graph display the size of each preparation (horizontal axis, in bp) and the respective intensity of each peak (vertical axis, FU). Expected small RNA library peaks around 120-160 bp, as seen in the figure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/67379fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67379/67379fig05.jpg" /> 
Figure 5: Quality check for miRnome sequencing. (A) FASTQC plot for read quality for the individual samples after adapter trimming. The horizontal axis is for nucleotide position, and the vertical axis is for Phred scores. Box plots for each position show the distribution of the Phred score across all reads for each position. All distributions within the green zone confirm very high-quality data (&#62;Q30) implicating high-quality sample preparation. (B) The sequence length distribution for each sample shows that for all three samples, the majority of the reads were around 21-22 nucleotides - the expected size range for microRNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/67379fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67379/67379fig06.jpg" /> 
Figure 6: Overlap of MicroRNA expression across all samples. The number of miRNAs detected from each sample (read count &#8805; 10) and specific microRNAs shared in more than one sample are shown in the Venn diagram. All raw and normalized reads are shown in Supplementary File 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/67379fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody Epitope</td>
			<td>Dilution Factor of Antibody&#160;</td>
		</tr>
		<tr>
			<td>Mouse Anti-CD9</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Mouse Anti-CD63</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Mouse Anti-CD81</td>
			<td>1:2000&#8239;</td>
		</tr>
		<tr>
			<td>Mouse Anti-Flotillin 1</td>
			<td>1:2000&#8239;</td>
		</tr>
		<tr>
			<td>Mouse Anti-TSG101</td>
			<td>1:1000&#8239;</td>
		</tr>
		<tr>
			<td>Mouse Anti-Calnexin</td>
			<td>1:10 000&#8239;</td>
		</tr>
		<tr>
			<td>Anti-mouse Secondary Antibody</td>
			<td>1:3000&#8239;</td>
		</tr>
	</tbody>
</table>
Table 1: Antibody epitopes used for the characterization of BDsEVs and their respective dilution factors.&#8239; 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample&#8239;&#160;</td>
			<td>miRNA Concentration (ng/&#956;L)&#8239;&#160;</td>
		</tr>
		<tr>
			<td>1</td>
			<td>8.76</td>
		</tr>
		<tr>
			<td>2</td>
			<td>3.24</td>
		</tr>
		<tr>
			<td>3</td>
			<td>5.33</td>
		</tr>
	</tbody>
</table>
Table 2: Quantification data of isolated RNA.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample&#160;</td>
			<td>Aligned Sequences&#160;</td>
			<td>Mapped miRNA&#160;</td>
			<td>Percentage (%)&#160;</td>
		</tr>
		<tr>
			<td>Control Sample 1&#160;</td>
			<td>1 444 665</td>
			<td>1 133 437</td>
			<td>78.46</td>
		</tr>
		<tr>
			<td>Control Sample 2&#160;</td>
			<td>442 808</td>
			<td>381 631</td>
			<td>86.18</td>
		</tr>
		<tr>
			<td>Control Sample 3&#160;</td>
			<td>2 024 828</td>
			<td>1 983 726</td>
			<td>97.97</td>
		</tr>
		<tr>
			<td>Average&#160;</td>
			<td>1 304 100.33</td>
			<td>1 166 264.67&#160;</td>
			<td>89.43</td>
		</tr>
	</tbody>
</table>
Table 3: Amount of aligned and successfully mapped miRNA sequences. 
Supplementary File 1: Supplementary western blot analysis data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/Supplementary File 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Supplementary NTA control data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/Supplementary File 2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Raw and normalized read counts of miRNA expression analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/Supplementary File 3.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary File 4: Supplementary protein concentration data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67379/Supplementary File 4.pdf" target="_blank">Please click here to download this File.</a>

Harnessing the Power of MicroRNA Cargoes in Small Extracellular Vesicles Released from Fresh-Frozen Human Brain Sections

Here, we establish a protocol using minimal amounts of fresh-frozen brain tissue sections and an accessible high-speed centrifugation method coupled with size exclusion chromatography to obtain small extracellular vesicles as sources for microRNA (miRNA) biomarkers for neurological disorders.

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic ...

harnessing-power-microrna-cargoes-small-extracellular-vesicles

Nanyang Technological University

Research

JoVE Journal

Neuroscience

362 Views.  University of Salford. De meeste gezondheidsproblemen kunnen beter worden beheerd als ze vroeg worden ontdekt. Biomarkers die beschikbaar zijn in toegankelijke lichaamsvloeistoffen zijn de meest veelbelovende manieren voor vroege opsporing van gezondheidsproblemen. We rapporteren verbeteringen in de methoden voor het onderzoeken van microRNA-biomarkers die vrijkomen via kleine extracellulaire blaasjes uit de menselijke hersenen.Deze methode heeft ons in staat gesteld om vari...

Video: Benutting van de kracht van microRNA-ladingen in kleine extracellulaire blaasjes die vrijkomen uit vers ingevroren menselijke hersensecties

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Behavior

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetics

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Medicine

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Cancer Research

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Harnessing the Power of MicroRNA Cargoes in Small Extracellular Vesicles Released from Fresh-Frozen Human Brain Sections

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