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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	<li>Jia, Y., 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=Small+extracellular+vesicles+isolation+and+separation:+Current+techniques,+pending+questions+and+clinical+applications.">Small extracellular vesicles isolation and separation: Current techniques, pending questions and clinical applications.</a> Theranostics. 12 (15), 6548-6575 (2022).</li><li>Fornari, S., Sch&#228;fer, A., Jucker, M., Goriely, A., Kuhl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-like+spreading+of+Alzheimer's+disease+within+the+brain's+connectome.">Prion-like spreading of Alzheimer's disease within the brain's connectome.</a> J R Soc Interface. 16 (159), 20190356 (2019).</li><li>Zhang, 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=Tumor-derived+small+extracellular+vesicles+in+cancer+invasion+and+metastasis:+molecular+mechanisms,+and+clinical+significance.">Tumor-derived small extracellular vesicles in cancer invasion and metastasis: molecular mechanisms, and clinical significance.</a> Mol Cancer. 23 (1), 18 (2024).</li><li>Constantinides, V., 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=CSF+A&#946;42+and+A&#946;42/+A&#946;40+ratio+in+Alzheimer's+disease+and+frontotemporal+dementia.">CSF A&#946;42 and A&#946;42/ A&#946;40 ratio in Alzheimer's disease and frontotemporal dementia.</a> Diagnostics. 13 (4), 783 (2023).</li><li>Cheng, L., 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=Prognostic+serum+miRNA+biomarkers+associated+with+Alzheimer's+disease+shows+concordance+with+neuropsychological+and+neuroimaging+assessment.">Prognostic serum miRNA biomarkers associated with Alzheimer's disease shows concordance with neuropsychological and neuroimaging assessment.</a> Mol Psychiatry. 20 (10), 1188-1196 (2015).</li><li>Roser, A. E., Caldi Gomes, L., Sch&#252;nemann, J., Maass, F., Lingor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+miRNAs+as+diagnostic+biomarkers+for+Parkinson's+disease.">Circulating miRNAs as diagnostic biomarkers for Parkinson's disease.</a> Front Neurosci. 12, 625 (2018).</li><li>Jackson, N., Guerrero-Mu&#241;oz, M. J., Castillo-Carranza, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prion-like+transmission+of+tau+oligomers+via+exosomes.">The prion-like transmission of tau oligomers via exosomes.</a> Front Aging Neurosci. 14, 974414 (2022).</li><li>Vella, L., 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+rigorous+method+to+enrich+for+exosomes+from+brain+tissue.">A rigorous method to enrich for exosomes from brain tissue.</a> J Extracell Vesicles. 6 (1), 1348885 (2017).</li><li>Yousif, G., Qadri, S., Parray, A., Akhthar, N., Shuaib, A., Haik, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+derived+neuronal+markers:+Immunoaffinity+isolation+and+characterization.">Exosomes derived neuronal markers: Immunoaffinity isolation and characterization.</a> Neuromolecular Med. 24 (3), 339-351 (2022).</li><li>Kumar, K., 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=Recent+advances+in+microfluidic+approaches+for+the+isolation+and+detection+of+exosomes.">Recent advances in microfluidic approaches for the isolation and detection of exosomes.</a> TRAC-Trend Anal Chem. 159, 116912 (2023).</li><li>Ransom, L., 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=Human+brain+small+extracellular+vesicles+contain+selectively+packaged,+full-length+mRNA.">Human brain small extracellular vesicles contain selectively packaged, full-length mRNA.</a> Cell Rep. 43 (4), 114061 (2024).</li><li>Gomes, 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=A+novel+isolation+method+for+spontaneously+released+extracellular+vesicles+from+brain+tissue+and+its+implication+for+stress-driven+brain+pathology.">A novel isolation method for spontaneously released extracellular vesicles from brain tissue and its implication for stress-driven brain pathology.</a> Cell Commun Signal. 21 (1), 35 (2023).</li><li>Welsh, 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=Minimal+information+for+studies+of+extracellular+vesicles+(MISEV+2023):+From+basic+to+advanced+approaches.">Minimal information for studies of extracellular vesicles (MISEV 2023): From basic to advanced approaches.</a> J Extracell Vesicles. 13 (2), e12404 (2023).</li><li>Th&#233;ry, C., 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=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> J Extracell Vesicles. 7 (1), 1535750 (2018).</li><li>Pirisinu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long+journey+of+extracellular+vesicles+towards+global+scientific+acclamation.">The long journey of extracellular vesicles towards global scientific acclamation.</a> Adv Pharm Bull. 13 (3), 489-501 (2023).</li><li>Bender, A., Sullivan, B. P., Lillis, L., Posner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+and+chemical-based+methods+to+inactivate+endogenous+blood+ribonucleases+for+nucleic+acid+diagnostics.">Enzymatic and chemical-based methods to inactivate endogenous blood ribonucleases for nucleic acid diagnostics.</a> J Mol Diagn. 22 (8), 1030-1040 (2020).</li></ol>

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 fr...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.&#160;</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 &#956;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...

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 ...

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Research

JoVE Journal

Neuroscience

337 Views.  University of Salford. 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. 

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...