The differentially expressed miRNAs were validated by quantitative reverse transcription-PCR (RT-qPCR). EV samples were spiked with 1 pmol cel-miR-39-3p (RiboBio Co., Ltd.) after being fully lysed by MagZol. RNA were reverse transcribed for cDNA synthesis using Evo M-MLV RT Kit for qPCR (Accurate Biotechnology). qPCR was performed using SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology) on a CFX96 Real-Time PCR System (Bio-Rad). The relative expression of miRNAs was calculated by 2−ΔΔCt method with cel-miR-39-3p as an external reference. All the bulge-loop miRNAs RT primers and qPCR primers were purchased from RiboBio Co., Ltd. (Guangzhou, China).
Cel mir 39 3p
Manufactured by RiboBio
Sourced in China
Cel-miR-39-3p is a synthetic single-stranded RNA molecule that corresponds to a microRNA sequence from the nematode Caenorhabditis elegans. It is commonly used as a spike-in control for normalization in RNA extraction and quantification experiments, particularly in the context of microRNA analysis.
Automatically generated - may contain errors
Lab products found in correlation
Evo m mlv rt kit for qpcr, by Accurate Biology (1 mentions)
Sybr green premix pro taq hs qpcr kit, by Accurate Biology (1 mentions)
Cfx96 real time pcr system, by Bio-Rad (1 mentions)
Midetect a track mirna qrt pcr starter kit, by RiboBio (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Mirneasy serum plasma kit, by Qiagen (1 mentions)
Sybr green real time pcr master mix, by Vazyme (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Glycogen, by Thermo Fisher Scientific (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using cel mir 39 3p
1
Validating Differentially Expressed miRNAs
2
Quantification of Serum Exosomal miRNAs
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Total RNA from the serum exosomes was isolated by using the miRNeasy Serum/Plasma kit (Qiagen, Germany) according to the manufacturers' protocol. To normalize RNA content, 1 pmol of exogenous cel-miR-39-3p (RiboBio, China) was added into per 100 μg exosomes diluted in the QIAzol lysis reagent. The expression levels of the eight selected miRNAs were detected by using miDETECT A Track™ miRNA qRT-PCR Starter Kit (RiboBio, China) with Bio-Rad CFX96 Real-Time System. The cycling parameters of the qPCR program were as follows: 95°C for 10 min (enzyme activation), followed by 45 cycles of 95°C for 2 s (denaturation), 60°C for 20 s (annealing), and 70°C for 10 s (extension). Primer sequences used for qPCR amplification of all the miRNAs were purchased from RiboBio. The miRNAs expression levels were normalized to cel-miR-39-3p and expressed as log10 (2−[CT (miR)–CT (cel-miR−39-3p)]). For all qPCR experiments, the samples were run in quadruplicate.
3
Bovine Blood Exosome RNA Quantification
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Total RNA was extracted from bovine blood-derived exosomes by TRIzol (Invitrogen), and 500 ng total RNA was reverse transcribed using a kit (Vazyme, Nanjing, China) to obtain cDNA. Primer Premier 5.0 was used to design quantitative primers for differentially expressed transcripts between the D30 and D250 groups. Primer sequences are listed in Table S1A . The external reference was cel-miR-39-3p (RiboBio, Guangzhou, China). The reaction mixture comprised 1 μL cDNA, 0.5 μL of upstream and downstream primers, 10 μL SYBR Green Real-Time PCR Master Mix (Vazyme, Nanjing, China), and 8 μL ddH2O. The cycling conditions were as follows: pre-denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 40 s. After amplification, the dissociation curve was analyzed. Relative quantification was performed using the 2−ΔΔCT method.
4
Plasma RNA Isolation Protocol
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All blood samples were immediately collected upon admission to the hospital and before any therapy was administered. Whole blood was drawn into EDTA-K2 plasma tubes and centrifuged at 2000 × g for 10 minutes at 4 °C. Then, the plasma was saved and stored at −80 °C in individual RNAase-free Eppendorf tubes until RNA extraction. Total RNA was isolated from plasma with TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Synthetic Caenorhabditis elegans miR-39-3p (cel-miR-39-3p, RiBoBio, Guangzhou, China) was used as an exogenous reference gene to normalize sample-to-sample variation. To increase the yield of RNA isolation from plasma, glycogen (Invitrogen) at a final concentration of 100 μg/mL was added per sample during RNA precipitation. The purity and concentration of the total RNA were determined using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA).
5
Serum RNA Isolation for miRNA Analysis
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After centrifugation of peripheral venous blood at 2,860 × g for 10 min, serum was collected and subsequently stored at −80°C. Total RNA was isolated from 250 µl serum using traditional TRIzol® LS reagent (Invitrogen/Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. An aliquot of 1 µl cel-miR-39-3p (Guangzhou RiboBio Co., Ltd.) at a concentration of 1 µM was added to each sample to act as the external reference during RNA isolation.
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