Total miRNA of exosomes was extracted by miRNeasy Mini kit (Qiagen, MD, USA). A miRNA First Strand cDNA Synthesis kit (Tailing Reaction) (Sangon Biotech, Shanghai, China) was used to synthesize miRNA cDNA from the total miRNA of exosomes. The synthesized exogenous reference cel‐miR‐39‐3p standard RNA (1 pmol per sample; RiboBio, Guangzhou, China) was added to the total RNA of exosomes in advance. Real‐time PCR for miRNA was carried out using a MicroRNA qPCR Kit (SYBR Green Method) (Sangon Biotech). The miRNA levels in exosomes were normalized against the exogenous reference cel‐miR‐39. Sequences of primers (Sangon Biotech) used for real‐time PCR in this study are listed in Table 4 . The miR‐29a、miR‐149‐3p and miR‐3934‐5p as well as miRctrl mimics (2 μM per sample) were synthesized and transfected with lipofectamine RNAiMAX (Thermo Scientific, Rockford, IL, USA). The sequences were presented in Table 5 .
Cel mir 39 3p standard rna
Manufactured by RiboBio
Sourced in China
Cel-miR-39-3p standard RNA is a synthetic RNA molecule that corresponds to the sequence of the Caenorhabditis elegans microRNA cel-miR-39-3p. This standard RNA can be used as a reference for the quantification of cel-miR-39-3p in samples.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Microrna qpcr kit sybr green method, by Sangon (1 mentions)
Mirna first strand cdna synthesis tailing reaction kit, by Sangon (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Sbi exoquick tc ultra ev isolation kit, by System Biosciences (1 mentions)
Faststart universal sybr green master rox kit, by Roche (1 mentions)
Bca protein concentration assay kit, by Boster Bio (1 mentions)
Paraformaldehyde (pfa), by Sinopharm (1 mentions)
Phosphate buffer saline (pbs), by Sangon (1 mentions)
U6 primer, by RiboBio (1 mentions)
Transscript all in one first strand cdna synthesis supermix kit, by Transgene (1 mentions)
Perfectstart green qpcr supermix kit, by Transgene (1 mentions)
Lightcycler 96 instrument, by Roche (1 mentions)
Midetect a track mirna qrt pcr starter kit, by RiboBio (1 mentions)
4 protocols using cel mir 39 3p standard rna
1
Exosomal miRNA Extraction and Quantification
2
Exosome Isolation and RNA Analysis
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Exosomes in osteoblast cell culture supernatant were isolated using an SBI ExoQuick-TC ULTRA EV Isolation Kit (System Biosciences, USA). Separation and reverse transcription of exosomes into cDNA were performed by using an SBI Seramir Exosome RNA Amplification Kit (System Biosciences, USA) and Cel-miR-39-3p standard RNA (RiboBio, Guangzhou, China) as external references. RT-PCR was performed to detect RNA expression in exosome using a FastStart Universal SYBR Green Master (ROX) kit (Roche, Germany).
3
Reagents and Materials for Molecular Experiments
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In this experimental study, phosphate buffer saline (PBS) was purchased from Sangon
Biotech Co., Ltd (Shanghai, China) Paraformaldehyde (4%) was obtained from China National
Pharmaceutical Group Corporation (Shanghai, China). The BCA Protein Concentration Assay
Kit was purchased from Boster Biological Technology Co. Ltd (Wuhan, China). RNAiso Plus
(TRIzol) and PrimeScriptTM II 1st Strand cDNA Synthesis Kit were purchased from
Takara Biomedical Technology (Beijing) Co., Ltd (Beijing, China). The cel-mir-39-3p
standard RNA was obtained from Guangzhou RiboBio Co., Ltd (Guangzhou, China).
Biotech Co., Ltd (Shanghai, China) Paraformaldehyde (4%) was obtained from China National
Pharmaceutical Group Corporation (Shanghai, China). The BCA Protein Concentration Assay
Kit was purchased from Boster Biological Technology Co. Ltd (Wuhan, China). RNAiso Plus
(TRIzol) and PrimeScriptTM II 1st Strand cDNA Synthesis Kit were purchased from
Takara Biomedical Technology (Beijing) Co., Ltd (Beijing, China). The cel-mir-39-3p
standard RNA was obtained from Guangzhou RiboBio Co., Ltd (Guangzhou, China).
4
Exosomal miRNA and mRNA Quantification
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Total RNA of cells and exosomes was extracted by TRIzol (Thermo Fisher Scientific). When the exosomes were fully lysed, 1 pmol of Cel-miR-39-3p standard RNA was added. For miRNA detection, total RNA was reverse transcribed and amplified using the miDETECT A TrackTM miRNA qRT-PCR Starter Kit (RiboBio). For mRNA detection, total RNA was reverse transcribed with a TransScript All-in-One First Strand cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China). PerfectStart Green qPCR SuperMix kit (TransGen) was used for quantitative real-time PCR (qPCR) on a LightCycler 96 instrument (Roche, Basel, Switzerland). The relative quantification was carried out by the 2−ΔΔCT method. Cel-miR-39-3p was used as an external control for the detection of exosomal miRNAs, and U6 and β-actin were used as internal controls for cellular miRNA and mRNA detection, respectively. Cel-miR-39-3p standard RNA (Cat. miRB0000010), cel-miR-39-3p primer (Cat. miRA0000010) and U6 primer (Cat. miRAN0002) were purchased from RiboBio. Primer sequences for qRT-PCR are listed in Table S1 .
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