Total RNA (100 ng) was used to quantify mmu-mmiR-673–5p levels. RNA was first converted to cDNA using miRCURY LNA RT kit (Qiagen). cDNA was diluted 1/25 for RT-qPCR using miRCURY LNA SYBR Green kit and amplified using mmu-mmiR-673–5p specific primers (Qiagen) and U6 as a loading control. Quantitative PCR was carried out on a Roche LC480 light cycler and analysed using the second derivative method.
Mircury lna sybr green kit
Manufactured by Qiagen
The MiRCURY LNA SYBR Green kit is a laboratory equipment product designed for the detection and quantification of microRNA (miRNA) expression using real-time PCR technology. The kit includes reagents and components necessary for the sensitive and specific analysis of miRNA levels in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Mircury lna rt kit, by Qiagen (2 mentions)
Trizol ls, by Thermo Fisher Scientific (2 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Lc480 light cycler, by Roche (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Unisp6, by Qiagen (1 mentions)
Mirneasy serum plasma kit, by Qiagen (1 mentions)
Mirneasy serum plasma spike in control, by Qiagen (1 mentions)
5 protocols using mircury lna sybr green kit
1
Quantification of miR-673-5p Expression
2
Quantifying Mature miRNA Levels in Endothelial Cells
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For quantification of mature miRNA levels, trizol purified RNA was used. cDNA was synthesized from 100 ng RNA using miRCury LNA RT-Kit (Qiagen) according to the manufacturer’s protocol (PCR System 9,700, GeneAmp, PE Applied Biosystems). Each cDNA was amplified using the miRCury LNA SYBR Green Kit (Qiagen). The reaction mix was first incubated at 95 °C for 2 min. Then, 40 cycles of 95 °C for 10 s and 56 °C for 1 min following by 95 °C for 1 s, 60 °C for 15 s and 95 °C for 1 s. Finally, 40 °C for 30 s. Assay ID used were: hsa-miR-146b-5p (204,553, Exiqon), hsa-miR-223-3p (YP00205986CC, Qiagen) and U6 snRNA (YP00203907CC, Qiagen). The expression level of these genes was evaluated by quantitative RT-PCR (Light cycler 96, Roche Diagnostics, Basel, Switzerland). The relative amount of each miRNA was calculated using the comparative threshold (Ct) method with ΔCt, Ct (miRNA)–Ct (U6 snRNA) in mature endothelial cells.
3
Quantitative Analysis of miRNA Expression
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Total RNA was extracted from cells with TRIzol as described above. RNA from cytoplasm lysate was extracted with TRIzol LS (Invitrogen, Cat. No. 10296028) and from nuclear pellet with TRIzol. The miRNAs were reverse transcribed using universal miRCURY LNA kit (Qiagen: 339340). The cDNA was further used for quantification of miRNAs following the protocol of miRCURY LNA SYBR Green kit (Qiagen: 339346). PCR was performed on a 7500 real-time PCR system (Applied Biosystems) using TaqMan Universal PCR Master Mix No AmpErase UNG (Applied Biosystems) with the primers and probes included in the TaqMan microRNA Assay kits (ThermoFisher, hsa-miR-210-3p: Assay ID: 000512; hsa-miR-181a-5p: Assay ID: 000480; hsa-miR-21-5p: Assay ID: 000397; hsa-miR-27a-3p: Assay ID: 000408). PCR reactions were done in triplicates at 55°C 2 min, 95°C 3 min and 95°C 30 s, 60°C 30 s for 40 cycles in an optical 96-well plate.
4
Validating Differential miRNA Levels in Serum
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The differentially expressed miRNAs were further validated using reverse transcription RT-qPCR. Briefly, total RNA was isolated from 200 μL serum by the miRNeasy serum/plasma kit (Qiagen, 217184) according to the manufacturer’s instructions. In addition, 3.5 μL miRNeasy serum/plasma spike-in control (Qiagen, 219610) at 1.6 × 108 copies/μL was added to each sample. The total RNA was reverse-transcribed using miRCURY LNA RNA kit (Qiagen, 339340) that generates universal cDNA templates for all miRNAs present in the sample. The synthetic spike-in (UniSp6, Qiagen, 339340) was added to each sample, and the reaction was performed in the GeneTouch thermal cycle (Bioer). Then, miRNA-specific quantification was performed using miRCURY LNA SYBR Green kit (Qiagen, 339347) according to the manufacturer’s instructions. The expression of target miRNAs was normalized to the cel-miR-39-3p synthetic spike-in added during total RNA extraction.
5
Quantification of miRNA expression
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Total RNA was extracted from cells with TRIzol as described above. RNA from cytoplasm lysate was extracted with TRIzol LS (Invitrogen, Cat. No. 10296028) and from nuclear pellet with TRIzol. The miRNAs were reverse transcribed using universal miRCURY LNA kit (Qiagen: 339340). The cDNA was further used for quantification of miRNAs following the protocol of miRCURY LNA SYBR Green kit (Qiagen: 339346). PCR was performed on a 7500 real-time PCR system (Applied Biosystems) using TaqMan Universal PCR Master Mix No AmpErase UNG (Applied Biosystems) with the primers and probes included in the TaqMan microRNA Assay kits (ThermoFisher, hsa-miR-210-3p: Assay ID: 000512; hsa-miR-181a-5p: Assay ID: 000480; hsa-miR-21-5p: Assay ID: 000397; hsa-miR-27a-3p: Assay ID: 000408). PCR reactions were done in triplicates at 55°C 2 min, 95°C 3 min and 95°C 30 s, 60°C 30 s for 40 cycles in an optical 96-well plate.
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