细胞外囊泡中miRNA的鉴定

Sajni  Josson; Murali Gururajan; Leland  W.K. Chung

doi:10.21769/BioProtoc.2139

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Peer-reviewed

miRNA Characterization from the Extracellular Vesicles

细胞外囊泡中miRNA的鉴定

Sajni Josson email

Murali Gururajan email

Leland W.K. Chung email

发布: 2017年02月20日第7卷第4期 DOI: 10.21769/BioProtoc.2139 浏览次数: 7836

评审: HongLok LungShalini Low-NamAnonymous reviewer(s)

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The authors used this protocol in:

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May 2015

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Abstract

Cancer cells and cancer associated stromal cells co-evolve secrete extracellular vesicles to the surrounding regions and regulate several processes involved in cancer metastasis. miRNAs have been known to be mediators of cancer progression and metastasis. miRNAs consist of short noncoding RNA. miRNAs are stable in extracellular fluids such as serum, plasma and urine. miRNAs are secreted by cells in normal and diseased conditions. miRNAs signatures have been identified specific to certain disease conditions. Therefore they are valuable biomarkers for different diseases. In our study we identified certain miRNAs, miR-409-3p and miR-409-5p, which were secreted by activated stromal fibroblast cells and were taken up by cancer cells to induce explosive tumor growth, through activation of epithelial to mesenchymal transition of cancer cells. Here we describe a procedure to determine miRNAs (miR-409-3p and miR-409-5p) in extracellular vesicles, which were secreted by prostate cancer stromal cells expressing miR-409. In this procedure, conditioned media from the stromal fibroblasts was used to extract the vesicular fraction. RNA was purified from the vesicular fraction, and specific miRNA was reverse transcribed and quantitated using real-time PCR assay.

Keywords: miRNA (miRNA)

Extracellular vesicles (细胞外囊泡)

Quantitative real-time PCR (定量实时PCR)

Cancer (癌症)

Cancer associated stroma (癌症相关基质)

Biofluids (生物流体)

Biomarkers (生物标志物)

Exosomes (外来体)

Background

miRNA are short non-coding RNA of 20-23 nucleotides in size. miRNAs have been detected in tissues and body fluids. miRNA expression levels have been determined using Northern blotting and quantitative real-time PCR. miRNA are powerful biomarkers for disease conditions. Under frozen conditions, they are stable in biofluids. Recent emphasis is on the miRNA in the vesicular fractions of cells and extracellular fluids (Josson et al., 2015). Extracellular vesicles (EV) contain different proteins, lipids, DNA, RNA and miRNA. EVs range from sizes of 30 nm to a few µm. EVs has been isolated using kits or differential centrifugation (Morello et al., 2013). In this study we describe the detection of miRNA in the vesicular fraction of activated stromal fibroblast cells. miRNAs are typically detected by Northern blot or quantitative real-time PCR assay (Josson et al., 2015). Using the recently available exosome purification kits we isolated exosomes and purified the miRNA from this fraction. The specific miRNA was reverse transcribed and quantitated using real-time PCR assay.

Materials and Reagents

6 well plates
6-well dish
MicroAmp^® optical 96-well reaction plate (Thermo Fisher Scientific, Applied Biosystems^TM)
MicroAmp^® optical adhesive tape (Thermo Fisher Scientific, Applied Biosystems^TM)
SON normal human stromal fibroblast cells expressing miR-409 miRNA
Penicillin-streptomycin (1%, stock: 10,000 U/ml)
Exosome depleted FBS media supplement (System Biosciences, catalog number: EXO-FBS-50A-1 )
Exo-Quick-TC (System Biosciences, catalog number: EXOTC10A-1 )
SeraMir exosome RNA purification kit (System Biosciences, catalog number: RA808A-1 )
Heat inactivated fetal bovine serum (Bio-Whittaker)
DMEM
F-12K
Sodium bicarbonate
Insulin
Triiodothyronine
Transferrin
Biotin
Adenine
dNTP mixture (Thermo Fisher Scientific, Applied Biosystems^TM)
M_MLV reverse transcriptase (Thermo Fisher Scientific, Applied Biosystems^TM)
10x RT buffer (Thermo Fisher Scientific, Applied Biosystems^TM)
RNase inhibitor (Thermo Fisher Scientific, Applied Biosystems^TM)
dH₂O (Thermo Fisher Scientific, Applied Biosystems^TM)
Taqman miRNA primer sets for hsa-miR-409-3p, hsa-miR-409-5p and RNU6B (Thermo Fisher Scientific, Applied Biosystems^TM)
Taqman universal PCR master mix, No AmpErase^® UNG (Thermo Fisher Scientific, Applied Biosystems^TM)
T-medium (see Recipes)
Reverse transcription mix for 1 reaction (see Recipes)
Real Time PCR reaction mixture for a single reaction (see Recipes)

Equipment

Centrifuge
Nanodrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: Nanodrop^TM 2000 / 2000c Spectrophotometer )
Applied Biosystems 7500 Real-Time PCR machine (Thermo Fisher Scientific, model: 7500 Real-Time PCR System )
PCR machine

Software

Taqman software
Graphpad Prism software

Procedure

English

中文翻译

文章信息

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如何引用

Josson, S., Gururajan, M. and Chung, L. W. (2017). miRNA Characterization from the Extracellular Vesicles. Bio-protocol 7(4): e2139. DOI: 10.21769/BioProtoc.2139.