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

TY - JOUR

T1 - Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

AU - Yan, Yan

AU - Chang, Chi Chih

AU - Venø, Morten T.

AU - Mothershead, Colin R.

AU - Su, Junyi

AU - Kjems, Jørgen

PY - 2019/3

Y1 - 2019/3

N2 - 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.

AB - 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.

KW - Exosomes

KW - Extracellular RNAs

KW - Extracellular vesicles

KW - Genetics

KW - Issue 145

KW - Mesenchymal stem cells

KW - MicroRNAs

KW - Next generation sequencing

KW - Ribonucleproteins

KW - Small RNA library construction

KW - Small RNAs

UR - http://www.scopus.com/inward/record.url?scp=85063713275&partnerID=8YFLogxK

U2 - 10.3791/58655

DO - 10.3791/58655

M3 - Journal article

C2 - 30907893

AN - SCOPUS:85063713275

SN - 1940-087X

VL - 2019

JO - Journal of visualized experiments : JoVE

JF - Journal of visualized experiments : JoVE

IS - 145

M1 - e58655

ER -