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

Summary

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.

Abstract

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 – 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.

Introduction

Extracellular and circulating RNAs (exRNAs) are present in various bodily fluids and are resistant towards RNases^1,2. Their high abundance, stability and ease of accessibility are attractive for clinical assessment as diagnostic and prognostic markers³. The mode of transport for exRNAs include extracellular vesicles (EVs), association with lipoproteins (such as high-density lipoprotein; HDL) and ribonucleoprotein complexes (such as with Argonaute2 complexes)⁴.

A subset of exRNAs are microRNAs (miRNAs), which are small non-coding RNAs of about 22 nt that regulate posttranscriptional gene expression. Ex-miRNAs have been implicated in cell-cell communication and regulation of cell homeostasis⁵. For example, HDL delivers ex-miR-223 to endothelial cells to repress intercellular adhesion molecule 1 (ICAM-1) and inflammation⁶. Interestingly, miR-223 is also seen transported by extracellular vesicles from leukocytes to lung cancer cells, programming them to take on a more invasive phenotype⁷. Thus, the transcriptome of ex-miRNAs from various bodily fluids and cell culture medium will greatly improve our understanding of ex-miRNA signaling.

Small RNA sequencing (small RNA seq) is a powerful tool that can be used to understand the transcriptomics of small RNAs. Not only can different samples be compared amongst differentially expressed known RNAs, but novel small RNAs can also be detected and characterized. Consequently, it is also a robust method to identify differentially expressed miRNAs under different conditions. However, one of the hurdles of small RNA seq is the difficulty in generating small RNA seq libraries from low exRNA input fluids like cerebrospinal fluids, saliva, urine, milk, serum and culture media. The TruSeq Small RNA Library Prep protocol from Illumina requires approximately 1 µg of high quality total RNA and the NEBNext Small RNA Library Prep Set protocol from New England Biolabs requires 100 ng-1 µg of RNA^8,9. Oftentimes, total RNA from these samples are below detection limit for conventional UV-vis spectrophotometers.

Ex-miRNAs derived from bodily fluids are potentially good prognostic and diagnostic markers. However, in order to study the functional effects or to determine the origin of specific ex-miRNAs, cell culture systems are often used instead. Mesenchymal stem cells (MSCs) have been studied extensively because their EVs have been implicated in alleviating many diseases including myocardial infarction, Alzheimer's disease and graft versus host disease¹⁰. Here, we demonstrate the purification of ex-miRNAs from bone marrow-derived MSCs (BMSCs) and the specific steps used to optimize small RNA library construction, sequencing and data analysis (Figure 1).

Protocol

NOTE: Mesenchymal stem cell growth medium (MSC media) is prepared beforehand as indicated in the Table of Materials.

1. Cell culture

NOTE: Human mesenchymal stem cells can be obtained from the bone marrow, adipose tissue or other sources¹¹. Alternatively, hMSCs can be bought through a supplier. The BMSCs used in this protocol were derived from the bone marrow of patients and bought from a company.

1. Thaw 1 x 10⁶ BMSCs into a T175 flask containing 20 mL of MSC media. Incubate the cells at 37 °C with 5% CO₂ and replace the media every 2-3 days until 80% confluency.
2. Wash the cells with 5 mL of 1x PBS and discard the PBS.
3. Detach the cells by adding 5 mL of 0.05% Trypsin-EDTA and incubating the cells for 5 min at 37 °C. Tap the sides of the flask to facilitate detachment.
4. Add 15 mL of MSC media to inactivate the trypsin, detach the cells from the surface, and pipette up and down to obtain single cell suspensions.
5. Collect the cells in a 50 mL tube and spin down for 5 min at 300 x g to pellet the cells.
6. Resuspend the cells in 1 mL of MSC media and count the cells using a hemocytometer.
   NOTE: Primary human bone marrow MSCs at 80% confluency in a T175 flask is around 2 x 10⁶ cells.
7. Plate hMSCs at 2,000 cells/cm² in fresh MSC media in 5 T175 flasks. Grow the cells at 37 °C with 5% CO₂ and replace the media every 2-3 days until 5 flasks of 90% confluent T175 flasks are obtained.

2. EVs and RNA-associated biomolecules collection

NOTE: EV collection media is prepared beforehand (Dulbecco's Modified Eagle's Medium [DMEM] with 10% fetal bovine serum [FBS] and 1% penicillin/streptomycin [P/S]). EV collection media is normal MSC media, but prepared with commercial EV-depleted FBS (Table of Materials). This is to avoid bovine exRNA contamination from FBS, which normally contains exRNAs associated with EVs, ribonucleoproteins, and lipoproteins. For small RNA library preparation, exRNAs derived from 5 confluent flasks of MSCs are required to enable library construction.

1. Wash the cells 3x with 20 mL of PBS per T175 flask. Add 20 mL of EV collection media per confluent T175 flask of MSCs and incubate at 37 °C with 5% CO₂ for 48 h.
2. Collect the media and centrifuge the media for 10 min at 300 x g and 4 °C.
3. Collect the supernatant and centrifuge the media for 20 min at 2,000 x g and 4 °C.
4. Collect the supernatant and centrifuge the media for 30 min at 15,500 x g and 4 °C. Then collect the supernatant.
5. Transfer the media to ultracentrifuge tubes and pellet the exRNAs for 90 min at 100,000 x g and 4 °C. A fixed angle rotor is used here and the pellet is anchored to the side of the tube. Mark the side of the lid and draw a circle on the side of the tube where the pellet is expected.
6. Remove the supernatant, dry the inside of the tube by inverting the tube on absorbent paper and use small pieces of absorbent paper to remove the liquid inside the tube without touching the bottom of the tube. Resuspend the pellet in 200 μL of PBS by vortexing for 30 s and pipetting up and down 20x.
7. Assess the EVs and biomolecules with nanoparticle tracking analysis (NTA) (Figure 2).
   NOTE: The EVs and biomolecules can be assessed with nanoparticle tracking analysis (NTA), dynamic light scattering (DLS) or transmission electron microscopy (TEM)¹².
8. Store the EVs and biomolecules at -80 °C until further downstream experiments.
   NOTE: If EVs are going to be used for functional studies, 20% glycerol must be added to protect them from rupturing. The cells can be collected using standard procedures if necessary.

3. EVs and RNA-associated biomolecules collection of differentiated cells

NOTE: EVs and RNA associated biomolecules can also be collected from the cell culture media while the cells undergo differentiation. The example depicted in the protocol describes osteoblastic differentiation and exRNA collection at day 0 and 7 of differentiation. If no differentiation is required, then skip Section 3 and go to Section 4.

1. Prepare osteoblastic differentiation media (DMEM with 10% FBS, 1% P/S, 10 mM β-glycerophosphate, 10 nM dexamethasone, 50 μM ascorbate-2-phosphate, and 10 mM 1.25-vitamin-D3) fresh every time.
2. Once MSCs are 80% confluent, change the MSC media to osteoblastic differentiation media.
3. Replenish the osteoblastic differentiation media after 2-3 days.
4. On day 5 of differentiation, remove the media and wash the cells 3x with 20 mL of PBS per T175 flask.
5. Add 20 mL of EV collection media containing 10 mM β-glycerophosphate, 10 nM dexamethasone, 50 μM ascorbate-2-phosphate and 10 mM 1.25-vitamin-D₃ per confluent T175 flask of MSCs. Incubate the cells at 37 °C with 5% CO₂ for 48 h to ensure continued differentiation while collecting the EVs and biomolecules.
6. Collect the media on Day 7 and proceed to isolate the EVs and biomolecules as described in steps 2.2-2.6.
7. For quality control, seed cells in 96-wells or 6-wells to assess differentiation using an alkaline phosphatase (ALP) activity assay or with quantitative polymerase chain reaction (qPCR), respectively.
   NOTE: Figure 3 is an example showing osteogenic differentiation of the cells.

4. RNA extraction and quality control

1. Thaw samples from step 2.8 on ice. Extract RNA using an RNA isolation kit (Table of Materials).
2. Elute the RNA from the column provided in the RNA isolation kit (Table of Materials) in 100 μL of RNase-free water.
3. Concentrate the RNA through ethanol precipitation by adding 1 μL of glycogen, 10 μL of 2 M pH 5.5 sodium acetate and 250 μL of pre-chilled 99% ethanol into 100 μL of purified RNA.
4. Incubate the samples at -20 °C overnight to precipitate the RNA. Pellet the RNA by centrifuging for 20 min at 16,000 x g and 4 °C.
   NOTE: The pellet is white due to co-precipitation with glycogen.
5. Remove the supernatant and wash the RNA pellet with 1 mL of 75% ethanol. Pellet the RNA again for 5 min at 16,000 x g and 4 °C.
6. Remove the ethanol and leave the lid of the RNA tube open for 5-10 min to air dry the RNA pellet. Resuspend the RNA pellet in 7 μL of RNase-free water.
7. Check the RNA quality and concentration using a chip-based capillary electrophoresis machine to detect the RNA according to the manufacturer’s protocol prior to library construction.
   NOTE: A representative size distribution of the RNA is shown in Figure 4.
8. Extract cellular RNA using a commercial purification kit (Table of Materials) if necessary.

5. Library construction and quality control

NOTE: Small RNA libraries are constructed using commercial kits (Table of Materials) with adjustments due to the low RNA input. Library construction is performed on the chilled block.

1. Chill the heating block for 0.2 mL PCR tubes on ice and pipette 5 μL of the RNA from step 4.6 into 0.2 mL RNase-free PCR tubes on a chilled block.
2. Dilute 3’ adaptors (1:10) in RNase-free water in a 0.2 mL RNase-free PCR tube. Add 0.5 μL of diluted adaptor and mix with 5 μL of RNA by pipetting up and down 8x and centrifuge briefly to collect all the liquid at the bottom of the tube.
3. Incubate the RNA and 3’ adaptor mix at 70 °C for 2 min in a preheated thermal cycler and then place the sample back on the chilled block.
4. Add 1 μL of ligation buffer, 0.5 μL of RNase inhibitor, and 0.5 μL of T4 RNA ligase (deletion mutant) into the RNA and 3’ adaptor mixture. Mix by pipetting up and down 8x and centrifuge briefly.
5. Incubate the tube at 28 °C for 1 h in the preheated thermal cycler.
6. Add 0.5 μL of stop solution into the sample tube with the tube staying in the thermal cycler, mix by pipetting up and down 8x and continue to incubate at 28 °C for 15 min.
7. Dilute 5’ adaptors (1:10) in RNase-free water in a 0.2 mL RNase-free PCR tube. Add 0.5 μL of 5’ adaptor into a separate RNase-free 0.2 mL PCR tube, heat the 5’ adaptor at 70 °C for 2 min in the preheated thermal cycler, and then place the sample on the chilled block.
8. Add 0.5 μL of 10 nM ATP, 0.5 μL of T4 RNA ligase to the 5’ adaptor tube, mix by pipetting up and down 8x and centrifuge briefly to collect all the liquids into the bottom.
   NOTE: Keep the 5’ adaptor on chilled block as much as possible.
9. Add 1.5 μL of the 5’ adaptor mixture to the sample from step 5.6, and mix very gently by pipetting 8x slowly. Incubate the sample at 28 °C for 1 h in the preheated thermal cycler.
10. Dilute RT primer 1:10 in RNase-free water in an RNase-free 0.2 mL PCR tube. Add 0.5 μL of diluted RT primer into the sample from step 5.9, mix very gently by pipetting up and down 8x slowly and centrifuge briefly.
11. Incubate the sample at 70 °C for 2 min in the preheated thermal cycler and then place the sample on a chilled block.
12. Add 1 μL of 5x first strand buffer, 0.5 μL of 12.5 mM dNTP mix, 0.5 μL of 100 mM dithiothreitol (DTT), 0.5 μL of RNase inhibitor, and 0.5 μL of reverse transcriptase. Mix very gently by pipetting up and down 8x slowly and centrifuge briefly.
13. Incubate the sample at 50 °C for 1 h in the preheated thermal cycler to obtain cDNA.
14. Add 4.25 μL of ultrapure water, 12.5 μL of PCR mix, 1 μL of index primer, and 1 μL of universal primer into the cDNA. Mix by pipetting up and down 8x and centrifuge briefly.
15. Place the sample in a thermal cycler and set up the cycler as follows: 98 °C for 30 s; 15 cycles of 98 °C for 10 s, 60 °C for 30 s and 72 °C for 15 s; and end the cycle with 72 °C for 10 min.
    NOTE: The prepared library can be stored at -20 °C for one week.
16. Purify the RNA library by separating the library on a DNA gel and cutting the gel (gel extraction) between 140 bp and 160 bp and eluting the library from the gel following the protocol given by the library construction kit.
    NOTE: In this study, the prepared library from step 5.15 is loaded onto a commercial gel (Table of Materials) and the library between 140 bp and 160 bp is purified out by an automated DNA size fractionator (Table of Materials) according to the manufacturer’s protocol.
17. Concentrate and wash the library from step 5.16 using a column-based PCR purification kit (Table of Materials) and elute the library in 10 μL RNase-free water finally.
18. Load 1 μL of the purified library onto a DNA chip (Table of Materials) to check the size of the library following the manufacturer’s protocol.
19. Dilute 1 μL of the purified library (1:1000) in 10 mM pH 8.0 Tris-HCl with 0.05% polysorbate 20 and quantify the library by qPCR using a commercial library quantification kit to quantify the library concentration using the DNA standards in the kit.
20. Pool equal amounts of the libraries according to the requirements of the sequencing machine and sequence on a high-throughput sequencing system.
    NOTE: The library construction of cellular RNA can be done using the same kits by following standard protocol of the kits.

6. Bioinformatics pipeline

NOTE: This is an in-house pipeline and the programs used here are listed in the Table of Materials.

1. Trim away low-quality reads and remove adaptor sequences from the raw reads.
2. Map the clean reads to different kinds of RNAs.
   1. Annotate tRNAs by allowing two mismatches because the heavily modified tRNA sequences cause frequent base misincorporation by reverse transcriptase.
   2. Annotate miRNAs by mapping to human miRNAs from miRBase v21 allowing zero mismatches. Since miRNAs are subject to A and U nontemplated 3’end additions, sequences that do not map are 3’trimmed of up to three A and/or T nts after which reads are mapped to human miRNAs and other miRNAs from miRBase v21 allowing zero mismatches.
   3. Annotate to other relevant small RNAs (snRNA, snoRNA, piRNA, and Y RNAs) by allowing one mismatch.
   4. Map the remaining unmapped reads to long RNA datasets (rRNA, other RNA from Rfam, and mRNA) to assess degradation.
   5. Annotate the sequences to the human genome.
   6. Annotate the sequences to bacterial genomes.
   7. Normalize miRNA expression using the following formula: miRNA counts / the total counts of all mapped miRNAs) x 10⁶.

Results

The method described in this protocol is optimized to collect exRNA from MSC culture for next generation sequencing. The overall scheme of the workflow is in Figure 1 on the left and the respective quality control checkpoints are on the right.

The morphology of the cells on the day of collection for undifferentiated (Figure 3A) and differentiated (F...

Discussion

Here, we describe a protocol for next generation sequencing of exRNAs that enables differential expression analyses from low input samples. Adhering to a specific protocol for EV and exRNA isolation is important because even small alterations (i.e., the ultracentrifugation step or a change in rotor type) can influence the transcriptome and miRNA levels^13,14. Thus, regardless of how the exRNA is isolated, it is important to apply the same experimental and bioinfor...

Materials

Name	Company	Catalog Number	Comments
Bone Marrow-Derived Mesenchymal Stem Cells	ATCC	PCS-500-012	Cells used in this protocol was bought from ATCC
MSCGM BulletKit	Lonza	PT-3001	Termed as Mesenchymal Stem Cell Growth Medium (MSC media)
Exosome-depleted FBS	Gibco	A2720801
ExRNA collecting media: MSCGM but with the FBS replaced by exosome-depleted FBS
Trypsin-EDTA	Gibco	25200056
T175 Flask	Sarstedt	833,912
Penicillin-Streptomycin	Gibco	15140122
Phosphate Buffered Saline	Sigma	806552
Ultracentrifuge	Beckman Coulter
Polycarbonate Bottle with Cap Assembly	Beckman Coulter	355618
Beckman Coulter Type 60 Ti			Rotor used here
NucleoCounter	Chemometec	NC-3000	Cell Counter
β-glycerophosphate	Calbiochem	35675	Components of the osteogenic differentiation media
Dexamethasone	Sigma	D4902-25MG	Components of the osteogenic differentiation media
2-Phospho-L-ascorbic acid trisodium salt	Sigma	49752-10G	Components of the osteogenic differentiation media
1α,25-Dihydroxyvitamin D3	Sigma	D1530-1MG	Components of the osteogenic differentiation media
miRNeasy Mini Kit	Qiagen	217004	miRNA and total RNA purification kit for step 4.8
Agilent RNA 6000 Pico Kit	Agilent Technologies	5067-1514	Chip-based capillary electrophoresis machine and chips for RNA and DNA analysis
Agilent 2100 Bioanalyzer	Agilent Technologies	G2939BA	Chip-based capillary electrophoresis machine and chips for RNA and DNA analysis
Agilent High Sensitivity DNA Kit	Agilent Technologies	5067-4626	Chip-based capillary electrophoresis machine and chips for RNA and DNA analysis
KAPA Library Quantification Kits	Roche	KK4824	Library quantification kit used here
TruSeq Small RNA Library Prep Kit -Set A (24 rxns) (Set A: indexes 1-12)	Illumina	RS-200-0012	Small RNA library prepartion kit used in this protocol - used in step 5
Pippin Prep	Sage Science		Automated DNA gel extractor used in this protocol; manual extraction can be done too
MinElute PCR Purification Kit	Qiagen	28004	PCR purification in step 5.17
FASTX_Toolkit	Cold Spring Harbor Lab		Trimming low-quality reads in step 6
cutadapt			Adaptor removal in step 6
Bowtie			Mapping of clean reads in step 6
Samtools			To make the expression profile in step 6
Bedtools			To make the expression profile in step 6