miScript PreAMP PCR Kit
For preamplification of cDNA from samples containing low RNA amounts prior to real-time PCR analysis of miRNA
The miScript PreAMP PCR Kit performs preamplification to enable use of the miScript PCR System for miRNA quantification with starting samples containing low RNA amounts. The miScript PreAMP PCR Kit uses highly multiplex, PCR-based preamplification of up to 400 miRNA-specific cDNA targets in one reaction. The miScript PreAMP PCR Kit, used with miScript PreAMP Primer Mixes, 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. This provides sufficient template for multiple Pathway-Focused miScript miRNA PCR Arrays or multiple replicates of a miRNome miScript miRNA PCR Array, depending on the miScript PreAMP Primer Mix used.
Highly significant increase in data from very limited sample amounts
The miScript PreAMP PCR Kit provides the opportunity to achieve large amounts of reliable miRNA profiling data from small samples and samples with very low RNA content. Previously unusable samples can now be used to elucidate valuable information about miRNA expression and function. Using preamplification, less than 1 µl serum equivalence RNA can be used to robustly profile miRNA expression using the Serum & Plasma miScript miRNA PCR Array (see figure "100% increase in miRNAs detected from 10-fold less cDNA after preamplification from serum").
High correlation with results from nonpreamplified samples
Reliable, unbiased amplification using the miScript PreAMP PCR Kit provides data that correlates highly with nonpreamplified sample, ensuring high confidence in results. Comparison of miRNA profiling results from tumor and normal lung tissue FFPE samples demonstrated this strong correlation (see figures "Preamplification preserves expression patterns in FFPE samples using 1000 fold less input cDNA" and "Extremely high correlation of expression between preamplified and nonpreamplified FFPE samples").
Reproducible results and robust procedure
Preamplification using the miScript PreAMP PCR Kit is a robust procedure that ensures consistent, reproducible results. Highly overlapping results were achieved when preamplification and miRNA profiling experiments were performed by different users (see figure "High technical reproducibility").
The miScript PCR System enables sensitive, specific miRNA quantification and profiling using SYBR® Green real-time PCR. The miScript PCR System covers all the steps involved in conversion of RNA to cDNA and subsequent real-time PCR detection of miRNAs. The miScript PreAMP PCR Kit is a component of the miScript PCR System that allows researchers to perform miRNA profiling experiments using very limited amounts of starting RNA (see Janas, M.M. et al. (2012) Reduced Expression of Ribosomal Proteins Relieves MicroRNA-Mediated Repression. Molecular Cell 46, 171.). This is necessary when working with samples that contain low amounts of RNA, such as body fluids, formalin-fixed, paraffin-embedded (FFPE) samples, and low cell number samples, such as laser capture microdissection (LCM) samples, flow-sorted cells, circulating tumor cells, and fine-needle biopsies. The low RNA yields obtained from small samples are often insufficient for reliable miRNA profiling experiments, even when using sensitive techniques such as real-time RT-PCR.
The miScript PreAMP PCR Kit, used with miScript PreAMP Primer Mixes, is a breakthrough technology enabling 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. This provides sufficient template for multiple Pathway-Focused miScript miRNA PCR Arrays or multiple replicates of a miRNome miScript miRNA PCR Array, depending on the miScript PreAMP Primer Mix used. The miScript PreAMP PCR Kit uses highly multiplex, PCR-based preamplification of up to 400 miRNA-specific cDNA targets in one reaction. Typically, preamplification results in a 2000–4000-fold amplification in either a 96-plex or 384-plex reaction.
miRNA profiling from low-quantity RNA samples requires the following 3 steps: reverse transcription using the miScript II RT Kit, preamplification using the miScript PreAMP PCR Kit and miScript PreAMP Primer Mix, and real-time PCR using the corresponding miScript miRNA PCR Array and the miScript SYBR Green PCR Kit. Preamplification using the miScript PreAMP PCR Kit and miScript PreAMP Primer Mix is therefore fully integrated into the miScript PCR System miRNA profiling workflow, ensuring robust protocols and reliable, reproducible results.
miScript PreAMP Primer Mixes are available for all miScript miRNA PCR Arrays. Alternatively, if desired, preamplification can be performed using the miScript PreAMP PCR Kit together with a mix of up to 25 miScript Primer Assays.
The miScript PreAMP PCR Kit includes 4 miScript Primer Assays for use in preamplification control experiments to test preamplified cDNA. Controls and their purpose are listed below. For more detailed information, see the miScript PreAMP Handbook.
The miScript PreAMP PCR Kit is used as part of the miScript PCR System for:
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