RNA enriched for small non-coding RNAs was extracted from sera (200 uL) using an miRNeasy serum kit (Qiagen). For normalization of sample-to-sample variation during RNA isolation procedures, synthetic C. elegans miRNA (cel-miR-39, Qiagen) was added to each denatured serum. miRNA expression was quantified using TaqMan miRNA real-time RT-qPCR assays (4427975) and QuantStudioTM 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA). The following primers (assay ID) were used for the OCaMIR signature RT-qPCR assay: hsa-miR-202–3p (#2363), hsa-miR-205–5p (#509), hsa-miR-508–3p (#1052), hsa-miR-509–3-5p (#2155), hsa-miR-513c-5p (#2756), hsa-miR-183–5p (#2269), hsa-miR-182–5p (#2334), and hsa-miR-513b-5p (#2757) (Thermo Fisher Scientific, Waltham, MA). Several endogenous controls including U6 (#1973), RNU44, hsa-miR-16–5p, and hsa-miR-103a-3p (Thermo Fisher Scientific) were tested for data normalization. All the endogenous controls were expressed without any difference between healthy controls and cancer specimens. We used U6 for data normalization of the Czech and West China cohorts. Expression of each miRNA was calculated using the 2-ΔCT method.
Mirneasy serum kit
Manufactured by Qiagen
Sourced in Germany
The MiRNeasy serum kit is a laboratory equipment product designed for the extraction and purification of microRNA (miRNA) from serum, plasma, and other liquid biological samples. The kit utilizes a proprietary technology to efficiently capture and isolate miRNA without the need for organic solvents or time-consuming procedures.
Automatically generated - may contain errors
Lab products found in correlation
Rnu44, by Thermo Fisher Scientific (2 mentions)
Ms2 carrier rna, by Roche (2 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Hsa mir 205 5p, by Thermo Fisher Scientific (1 mentions)
Cel mir 39, by Qiagen (1 mentions)
Sybr green pcr kit, by Qiagen (1 mentions)
Mirneasy ffpe kit, by Qiagen (1 mentions)
Lightcycler 480 rt pcr system, by Roche (1 mentions)
7 protocols using mirneasy serum kit
1
Quantification of Serum miRNA Expression
2
Serum and CSF miRNA Extraction Protocol
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RNA from 200 μl of thawed serum or CSF samples was isolated using the miRNeasy serum kit (Qiagen, Crawley, UK), according to the manufacturer's instructions. In order to increase RNA yield, we first added MS2 carrier RNA (Roche, Welwyn Garden City, UK) to QIAzol to give a final concentration of 1.25 μg ml−1 (Murray et al, 2015b (link)). To measure the RNA extraction efficiency, we also made a master mix of the exogenous non-human spike-in cel–miR–39–3p from the kit and added a fixed quantity (5.6 × 108 copies) to each sample (Murray et al, 2014 (link)), as per the manufacturer's instructions. This fixed quantity resulted in subsequent cel–miR–39–3p qRT–PCR levels within the expected range for endogenous human serum miRNA levels (Murray et al, 2014 (link)). For samples where 200 μl was not available, volumes were made up to 200 μl with an appropriate volume of 1 × PBS. Total RNA was eluted from columns with 100 μl of nuclease-free water and stored at −80 °C.
3
Serum miRNA Quantification Protocol
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RNA was extracted from serum with the miRNeasy Serum Kit (Qiagen, USA) according to the manufacturer's protocols. The concentration and purity of extracted RNA were evaluated by Nano Drop2000 (Thermo Scientific, USA) based on the value of OD260/280.
RNA was reverse transcribed into cDNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). cDNA was amplified with the SYBR Green PCR Kit (Qiagen, USA). Relative expression was calculated with the 2−ΔΔCt method normalized to GAPDH.
RNA was reverse transcribed into cDNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). cDNA was amplified with the SYBR Green PCR Kit (Qiagen, USA). Relative expression was calculated with the 2−ΔΔCt method normalized to GAPDH.
4
Serum microRNA Extraction and Profiling
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For all analyses, RNA was extracted from 200 μL of serum using the miRNeasy serum kit (Qiagen) into a final volume of 100 μL nuclease free water as described previously (12 (link)). All isolated RNA was stored at -80°C. RNA yield was maximised with the addition of MS2 carrier RNA (Roche, final concentration 1.25 μg ml−1) to Qiazol prior to isolation and the exogenous spike-in miRNA cel-miR-39-3p (5.6 × 108 copies) was used as an initial quality control for extraction efficiency. All samples underwent quality control analysis prior to subsequent target miRNA qRT-PCR analyses (12 (link), 16 (link)). Quality control analysis included quantification of cel-miR-39-3p, the endogenous housekeeper miR-30b-5p and the haemolysis control miRNAs miR-451a and miR-23a-3p as previously described (12 (link)). Consistency of extraction was acceptable for all samples analysed (14 (link)). Haemolysis assessment was performed by calculating the delta Ct values for miR-23a-3p minus miR-451a, detailed in (14 (link)).
5
FFPE Tissue and Serum miRNA Profiling
6
Serum RNA Isolation and Evaluation
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Total RNA extraction from collected serum samples were performed with the miRNeasy serum kit (QIAGEN, Germany). Cells were lysed with Trizol reagent (Life Technology, USA) for RNA extraction. Isolated RNA was evaluated for purity and concentration by the value of OD260/280.
7
Quantifying FLVCR1-AS1 Expression
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To investigate FLVCR1-AS1 expression in tissue samples and serums, qRT-PCR was applied on the Roche Lightcycler 480 RT-PCR system. The extraction of total RNA from tissue and cell samples was performed using TRIzol reagent (Invitrogen, Carlsbad, CA), while RNA in serum was done with Qiagen miRNeasy Serum Kit (Hilden, Germany). Then RNA samples were used for synthesis of cDNA. All of the specimens were tested in triplicate.
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