Validation of miRNA differential expression was performed using qRT-PCR on plasma samples from persons unrelated to those examined using NGS. RNA was isolated from 200 ul of plasma by miRCURY RNA Isolation Kits-Biofluids (Exiqon) and reverse transcribed by Universal cDNA Synthesis kit II (Exiqon). 7.5×10-2 fmol Serum/Plasma Spike-in Control (Qiagen) was added to each sample according to the manufacturer's protocol. The cDNA samples were added into Pick-&-Mix microRNA PCR Panels (Exiqon), which were pre-loaded with LNA PCR primers of miRNAs and inter-plate calibrator (UniSp3). The panels were run on a Stratagene Mx3005p machine. The expression of miRNAs was normalized to the Spike-in.
Mircury rna isolation kits biofluids
Manufactured by Qiagen
Sourced in Denmark
The MiRCURY RNA Isolation Kits-Biofluids are a series of kits designed for the extraction and purification of total RNA, including small RNAs such as miRNA, from various biofluid samples, including plasma, serum, urine, and cerebrospinal fluid.
Automatically generated - may contain errors
Lab products found in correlation
Universal cdna synthesis kit 2, by Qiagen (1 mentions)
Pick mix microrna pcr panel, by Qiagen (1 mentions)
Cfx384 real time system, by Roche (1 mentions)
Rna spike in kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Abi 7900t pcr system, by Thermo Fisher Scientific (1 mentions)
Tri reagent solution, by Merck Group (1 mentions)
4 protocols using mircury rna isolation kits biofluids
1
Plasma miRNA Validation by qRT-PCR
2
Extracellular Vesicle RNA Profiling
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Before inclusion into the study, serum samples were evaluated for the presence of hemolysis using a NanoDrop (hemolysed samples show a peak at 414 nm). RNA was purified with miRCURY™ RNA Isolation Kits-Biofluids (Exiqon) from resuspended EV samples or total serum samples. Briefly, 75 µl of lysis solution containing 1.25 µl of RNA spike-in templates UniSp2, UniSp4 and UniSp5 was added to samples. Protocol for RNA extraction was followed according to manufacturer's instructions. For the reverse transcription reaction with miRCURY RT kit (Exiqon), a volume of 2 µl of RNA per 10 µl of reaction was included as well as 0.5 µl of spike-in UniSp6 and cel-miR-39-3. Thus, UniSp2 and cel-miR-39-3 were used as RNA extraction and cDNA synthesis controls, respectively. After cDNA was diluted to 1:40, individual primer sets for UniSp2, cel-39-3, miR-16-5p, miR-21, miR-19a, miR-451, miR-30c, miR-143, miR-126 and miR-191 (all from Exiqon) were used and PCR was carried out in 384-well plates in a CFX384 Real-Time System (Roche).
3
Serum Total RNA Isolation and Spike-in
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Total RNA was extracted from 500 µL serum samples using miRCURY RNA Isolation Kits-Biofluids (Exiqon, Vedbaek, Denmark). Exiqon’s RNA spike-in kit for quality control of the RNA isolation was applied. Three RNA isolation controls (UniSp2, UniSp4 and UniSp5) pre-mixed, each at different concentration in 100 fold increments were added to the purification to detect any differences in extraction efficiency. We observed an excellent correlation of counts corresponding to the spike-ins between the samples.
4
Extracellular Vesicle RNA Profiling
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RNA was extracted using TRI reagent solution (Sigma) and RNeasy mini kit (Qiagen, Germany). RNA was also purified with miRCURY RNA Isolation Kits-Biofluids (Exiqon) from re-suspended EVs. Complementary DNA (cDNA) synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche for mRNA and Novabio for miRNA). qPCR assay was performed using ABI 7900 T PCR System (Applied Biosystems, Foster City, USA). The sequences of the primers used for detection are provided in Additional file 1 : Table S1.
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