miScript Microfluidics PCR Kit
For real-time PCR profiling of miRNAs using the Fluidigm BioMark System
The miScript Microfluidics PCR Kit performs miRNA quantification on the Fluidigm BioMark System with samples that have been preamplified using the miScript Microfluidics PreAMP Kit. The miScript PCR System for microfluidics represents a breakthrough technology enabling accurate and comprehensive miRNA expression analysis on the Fluidigm BioMark System with as little as 10 ng total RNA. Highly multiplex, PCR-based preamplification of up to 400 miRNA-specific cDNA targets in one reaction results in a 1000–4000-fold amplification of each target. Preamplification dramatically reduces the sample requirements for analysis. A single 1 ng cDNA synthesis reaction can be used as template for up to 10 preamplification reactions.
Reproducible results and robust proceduremiRNA profiling using the miScript Microfluidics PCR Kit, following preamplification, provides consistent, reproducible results in replicate experiments performed by different users. Data achieved correlates highly with nonpreamplified sample, ensuring high confidence in results.
The miScript PCR System for microfluidics allows researchers to perform miRNA profiling experiments using very limited amounts of starting RNA on the Fluidigm BioMark System. The Fluidigm BioMark System enables high-throughput experiments of up to 9216 (96 x 96) or 2304 (48 x 48) reactions on a single chip. The miScript PCR System for microfluidics is compatible with 96.96 or 48.48 Dynamic Array IFCs. This breakthrough technology enables accurate and comprehensive expression analysis with as little as 10 ng total RNA. A single 10 ng cDNA synthesis reaction can be used as template for up to 10 preamplification reactions. The miScript Microfluidics PreAMP Kit uses highly multiplex, PCR-based preamplification of up to 400 miRNA-specific cDNA targets in one reaction, resulting in a 1000–4000-fold amplification of each target. Preamplified sample is then used as template for miRNA profiling with the miScript Microfluidics PCR Kit.
miRNA profiling on the Fluidigm BioMark System requires the following 3 steps:
Reverse transcriptionTwo buffers, 5x miScript HiSpec Buffer and 5x miScript HiFlex Buffer, are provided in the miScript II RT Kit. miScript HiSpec Buffer facilitates the selective conversion of mature miRNAs into cDNA. miScript HiSpec Buffer is the only buffer that should be used to prepare cDNA for subsequent preamplification with the miScript Microfluidics PreAMP Kit. Do not use miScript HiFlex Buffer prior to preamplification with the miScript Microfluidics PreAMP Kit.
PreamplificationPreamplification using the miScript Microfluidics PreAMP Kit and an appropriate miScript PreAMP Primer Mix enables unbiased amplification of the miRNA targets of interest. The reaction includes a vital side reaction reduction step that minimizes any potential nonspecific reactions in real-time PCR. For each miScript miRNA PCR Array, there is a dedicated, verified miScript PreAMP Primer Mix that includes primers to selectively preamplify the miRNAs targeted by the array.
When real-time PCR will be performed with miScript Primer Assays, preamplification must be performed with a miScript PreAMP Primer Mix prepared by pooling the individual miScript Primer Assays.
miRNA profiling by real-time PCRPreamplified cDNA serves as the template for real-time PCR analysis using mature miRNA-specific forward primers (either in miScript miRNA PCR Arrays or as individual miScript Primer Assays) and the miScript Microfluidics PCR Kit, which contains the miScript Microfluidics Universal Primer (reverse primer), and Microfluidics qPCR Master Mix (which is optimized for the Fluidigm BioMark System and contains EvaGreen dye for detection and ROX passive reference dye). miScript miRNA PCR Arrays (Format M) are specifically designed for 96.96 Dynamic Array IFCs. This leading miRNA expression profiling technology contains miRNA-specific forward primers pre-dried at a concentration suitable for use with the Fluidigm BioMark System.
The miScript Microfluidics PCR Kit enables high-throughput miRNA expression profiling with the Fluidigm BioMark System for:
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