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 o....

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....

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

List of materials used in this article

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