miScript PreAMP PCR Kit

For preamplification of cDNA from samples containing low RNA amounts prior to real-time PCR analysis of miRNA

  • Breakthrough technology for miRNA expression analysis from limited RNA samples
  • Unbiased, reproducible results
  • Robust procedure
  • Fully integrated into the miScript PCR System workflow

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.

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miScript PreAMP PCR Kit (12)
For 12 preamplification reactions: 5x miScript PreAMP Buffer, HotStarTaq DNA Polymerase (2 U/µl), miScript PreAMP Universal Primer, miR-16 miScript Primer Assay, SNORD95 miScript Primer Assay, miRNA reverse transcription control (miRTC), C. elegans miR-39 miScript Primer Assay, RNase-Free Water
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miScript PreAMP PCR Kit (60)
For 60 preamplification reactions: 5x miScript PreAMP Buffer, HotStarTaq DNA Polymerase (2 U/µl), miScript PreAMP Universal Primer, miR-16 miScript Primer Assay, SNORD95 miScript Primer Assay, miRNA reverse transcription control (miRTC), C. elegans miR-39 miScript Primer Assay, RNase-Free Water
 331452
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The miScript PreAMP PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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100% increase in miRNAs detected from 10-fold less cDNA after preamplification from serum.|High technical reproducibility.|Preamplification preserves expression patterns in FFPE samples using 1000-fold less input cDNA.|Extremely high correlation of expression between preamplified and nonpreamplified FFPE samples.|
Total RNA was purified from 5 µl human serum using the miRNeasy Serum/Plasma Kit. cDNA was then prepared from 0.7 µl serum equivalence (SE) using the miScript II RT Kit with miScript HiSpec Buffer. cDNA (0.7 µl SE) was used directly for miRNA profiling or one-tenth of the cDNA preparation (0.07 µl SE) was preamplified using the miScript PreAMP PCR Kit with Serum & Plasma miScript PreAMP Pathway Primer Mix prior to profiling. miRNA profiling was performed with the with the Plasma & Serum miScript miRNA PCR Array. Plots of [A] mean CT values achieved and [B] number of miRNAs detected demonstrate highly superior results from 10 fold less starting cDNA due to preamplification.|cDNA was prepared from a human universal reference RNA sample using the miScript II RT Kit with miScript HiSpec Buffer. Three different users performed preamplification using the miScript PreAMP PCR Kit and miFinder miScript PreAMP Pathway Primer Mix. Preamplified cDNA was used for miRNA profiling in duplicate with the miFinder miScript miRNA PCR Array. High levels of correlation were observed between users when CT values were [A] compared on a scatter plot or [B] plotted for all 3 users.|Preamplified cDNA and nonpreamplified cDNA from the same prep were used for miRNA profiling. Scatter plots of x-fold expression change calculations (2-ΔΔCT) between normal and tumor sections demonstrate high correlation between nonpreamplified and preamplified samples. [A] 96-plex miFinder miScript PreAMP Pathway Primer Mix and Array or [B] 384-plex miScript PreAMP miRNome Primer Mix and Array were used. The miRNeasy FFPE Kit was used to purify RNA from normal and tumor lung tissue 5 µM FFPE sections. cDNA was prepared from 10 ng total RNA using the miScript II RT Kit with miScript HiSpec Buffer and preamplified using the miScript PreAMP PCR Kit.|Preamplified cDNA and nonpreamplified cDNA from the same prep were used for miRNA profiling. Scatter plots of ΔCT values (normalized against housekeeping controls) between normal and tumor sections demonstrate high correlation between nonpreamplified and preamplified samples. [A] 96-plex miFinder miScript PreAMP Pathway Primer Mix and Array or [B] 384-plex miScript PreAMP miRNome Primer Mix and Array were used. The miRNeasy FFPE Kit was used to purify RNA from normal and tumor lung tissue 5 µM FFPE sections. cDNA was prepared from 10 ng total RNA using the miScript II RT Kit with miScript HiSpec Buffer and preamplified using the miScript PreAMP PCR Kit.|
Performance
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").

Principle
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.
Procedure

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.

Preamplification controls

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.

Control Purpose
miR-16 miScript Primer Assay Determination of the optimal dilution factor for preamplified cDNA if the template starting amount is unknown
SNORD95 miScript Primer Assay Data normalization for tissue and cell samples using the ∆∆CT method of relative quantification
Determination of the optimal dilution factor for preamplified cDNA if the template starting amount is unknown and miR-16 is not present
C. elegans miR-39 miScript Primer Assay Measurement of miRNeasy Serum/Plasma Spike-In Control (if added during purification) for determination of recovery from serum and plasma samples
miRTC miScript Primer Assay Assessment of reverse-transcription efficiency

Applications

The miScript PreAMP PCR Kit is used as part of the miScript PCR System for:

Mature miRNA quantification and profiling
Biomarker discovery from serum or plasma
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Brochures & Guides
1
Total RNA Discovery - (EN)
Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow
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Kit Handbooks
3
(ZH) - miScript miRNA PCR Array操作手册

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(EN) - miScript miRNA PCR Array Handbook
For SYBR Green-based, real-time PCR profiling of microRNAs using pathway-focused arrays, HC arrays, and miRNome arrays
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(EN) - miScript PreAMP Handbook
For preamplification of cDNA from samples containing low RNA amounts prior to real-time PCR analysis of microRNA
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