RNA was isolated using the RNeasy Mini kit (Qiagen) in accordance with manufacturer’s instructions. cDNA was synthesized from equivalent concentrations of total RNA using the Super-Script III First-Strand Synthesis Super-Mix Kit (Invitrogen) in accordance with manufacturer’s instructions and the SimpliAmp thermal cycler systems (Applied Biosystems). Amplification was performed using a QuantStudio 3 Real-Time PCR System (Applied Biosystems). Cycle threshold values were determined and normalized to the loading control for each experiment. Fold changes for experimental groups relative to respective controls were calculated using MX Pro software (Agilent Technologies).
Simpliamp thermal cycler systems
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SimpliAmp thermal cycler systems are laboratory equipment designed for the amplification of DNA or RNA samples. The core function of the thermal cycler is to precisely control the temperature and duration of the heating and cooling cycles required for the polymerase chain reaction (PCR) process.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna assays kit, by Thermo Fisher Scientific (2 mentions)
Mirneasy serum plasma kit, by Qiagen (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis supermix kit, by Thermo Fisher Scientific (1 mentions)
Mxpro software, by Agilent Technologies (1 mentions)
3 protocols using simpliamp thermal cycler systems
1
Total RNA Extraction and qRT-PCR Analysis
2
Quantification of Plasma and Tissue miRNAs
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Total RNAs of plasma and tissue samples were extracted using QIAGEN miR-Neasy serum/plasma kit (QIAGEN) according to the manufacturer's instruction. Briefly, miRs were converted to cDNA by TaqMan MicroRNA Assays kit (Applied Biosystems, Waltham, Mass) in RT-PCR (reverse transcription polymerase chain reaction) machine (SimpliAmp Thermal Cycler systems, Applied Biosystems, Foster city, CA). All primers (mmu-miR-370-3p, ID 002275; mmu-miR-137-5p, ID 001129; mmu-let-7a-1-3p, ID 002478) were purchased from Thermo Fisher Scientific (Waltham, Mass) and cDNA samples for quantitative real-time PCR through TaqMan Universal PCR Master Mix on a qRT-PCR (real-time polymerase chain reaction) machine were used (Applied Biosystems). Relative expression was calculated using the ΔΔCT method and normalized to the expression of snoRNA-202 and cel-miR-39 for tissue and plasma samples, respectively (Applied Biosystems).
3
Extraction and Quantification of Serum and Tissue microRNAs
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Total RNAs of serum and tissue samples were extracted using QIAGEN miRNeasy serum/plasma kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The microRNAs (miRs) were converted to cDNA by TaqMan™ MicroRNA Assays kit (Applied Biosystems, Waltham, MA, USA) in RT-PCR (reverse transcription-polymerase chain reaction) machine (SimpliAmp™ Thermal Cycler systems, Applied Biosystems) using the mmu-miR370-3p primer (ID 002275) (Thermo Fisher Scientific) and cDNA samples for quantitative PCR through TaqMan™ Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA). Relative expression was calculated using the ∆∆CT method and normalized to the expression of snoRNA202 and cel-miR-39 for tissue and serum samples, respectively (Applied Biosystems). Because the miR results could be interfered by cell contamination and red blood cell rupture [86 (link)], sera were collected after centrifuged at 5000× rpm for 10 min before determination of hemolysis and RNA purity. Then, sample discoloration was excluded to reduce the interference from hemolysis. The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of RNA, and the RNA samples with an absorbance ratio (absorbance at 260/absorbance at 280) higher than 2.0 were used.
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