We isolated RNA and profiled 86 candidate miRNAs and 3 negative controls (SNORD61; SNORD95, RNU6-2 RNA), a Reverse Transcription Control, and a PCR Positive Control in miRhythm participants using Qiagen chemistry-based assays from tissue or venous blood samples. The BioMark System used to quantify miRNA levels has been validated extensively27 (link) and detects single miRNA copies at 26–27 Ct. Standard methods for cDNA conversion, pre-amplification, and qRT-PCR with Qiagen miScript Assays were used (Supplemental Tables 2–7, Supplemental Methods ). Since circulating miRNA levels can change within hours after organ injury,28 (link) we assessed miRNA levels within 3 hours of hospital presentation in participants with AF as well as in hospital controls.
Miscript assays
Manufactured by Qiagen
The MiScript Assays are a collection of pre-designed and validated assays for the detection and quantification of microRNAs (miRNAs) and other small non-coding RNAs. The assays are based on real-time PCR technology and provide a reliable and sensitive method for miRNA analysis.
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Lab products found in correlation
2 protocols using miscript assays
1
Profiling Circulating miRNAs in Atrial Fibrillation
2
Quantification of Nascent miRNAs
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Short factory RNA (<200 nt), isolated 0 or 30 min post-stimulation with TNFα as described above, was treated with RQ1 DNase (1 unit of DNase/μg of RNA; 37°C for 45 min; Promega) and precursor miRNAs amplified from nascent RNA using miScript assays (Qiagen) as per manufacturer's instructions on a Rotor-Gene 3000 cycler (Corbett). The presence of single amplimers was confirmed by melting-curve analysis; reactions in which the reverse-transcription step was omitted were performed to ensure amplimers did not result from residual genomic DNA and pre-miRNA levels were normalized relative to those of RNU6 snRNA.
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