RNeasy Serum/Plasma Kit (217184, Qiagen) was used to isolate the RNA from the EVs and HDL particles according to the manufacturer's instructions. Before isolation, 140 μL of filtered (0.2 μm) PBS (pH 7.4) was used to obtain the recommended starting volume of 200 μL. Spike‐in cel‐miR‐39‐3p (MS00019789, Qiagen) was used as an internal control (housekeeping miR).
Spike in cel mir 39 3p
Manufactured by Qiagen
Spike-in cel-miR-39-3p is a synthetic miRNA used as an internal control for quantification of miRNAs. It is a 22-nucleotide-long miRNA sequence derived from the Caenorhabditis elegans genome.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy serum plasma kit, by Qiagen (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Microrna assay kit, by Thermo Fisher Scientific (1 mentions)
Total exosome isolation kit, by Thermo Fisher Scientific (1 mentions)
Mirnaeasy kit, by Qiagen (1 mentions)
4 protocols using spike in cel mir 39 3p
1
RNA Isolation from Extracellular Vesicles
2
RNA Isolation from Extracellular Vesicles
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RNeasy Serum/Plasma Kit (217184, Qiagen) was used to isolate the RNA from the EVs and HDL particles according to the manufacturer's instructions. Before isolation, 140 µl of filtered (0.2 µm) PBS (pH 7.4) was used to obtain the recommended starting volume of 200µl. Spike-in cel-miR-39-3p (MS00019789, Qiagen) was used as an internal control.
3
Quantification of miR-148-3p Expression
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cDNA synthesis was performed with 500 ng of RNA using miScript II RT Kit (Qiagen), according to the manufacturer’s instructions. The cDNA product was then submitted to qPCR, in a 7900HT Fast Real-time PCR system (Applied Biosystems) with a 384 well plate format, using miScript SYBR Green PCR kit (Qiagen) and hsa-miR-148-3p specific oligo (Isogen LifeSciences, Utrecht, The Netherland) at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 sec and 58 °C for 1 min. Reactions were run in duplicate, miRNA expression was normalized with cel-miR-39-3p spike-in (Qiagen) or RNU6 cell housekeeping gene. Relative quantification was calculated by the 2−ΔΔCt method.
4
Exosome miRNA Isolation and Quantification
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Exosomes were isolated from the serum using a Total Exosome Isolation kit (Thermo Fisher Scientific, Gaithersburg, MD) according to the manufacturer’s instructions. Briefly, 50 µl of the serum was mixed with 10 µl of isolation reagent and incubated for 30 min at 4 °C. After centrifugation at 10,000 g for 10 min at 4 °C, the resulting pellet was dissolved in 100 µl of PBS, and miRNA was extracted using a miRNA easy kit (Qiagen, Hilden, Germany). Briefly, 500 µl of QIAzol reagent was added to 100 µl of isolated exosomes and incubated for 5 min at room temperature. After the addition of 5 µl of 5 nM Cel-miR-39-3p spike-in (Qiagen) and 100 µl of chloroform, samples were centrifuged at 12,000 g for 15 min at 4 °C. The upper aqueous phase was transferred to a fresh tube, and 1.5 volumes of ethanol were added. The sample was then applied to columns and washed. MiRNA was eluted in 30 µl of nuclease-free water. The quantity of miRNA was measured using Qubit (Thermo Fisher Scientific) and microRNA assay kit (Thermo Fisher Scientific).
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