miScript PreAMP Primer Mixes for miScript miRNA PCR Arrays
The miScript PreAMP PCR Kit, in combination with the miScript PreAMP Primer Mixes, enables use of the miScript PCR System for miRNA quantification with starting samples containing low RNA amounts. This is particularly important when working with samples such as body fluids, formalin-fixed, paraffin-embedded (FFPE) samples, and small cell number samples such as laser capture microdissection (LCM) samples, flow-sorted cells, circulating tumor cells, and fine needle biopsies, where the low RNA yields obtained are often insufficient for reliable miRNA profiling experiments. Normally, the low RNA yields obtained from such samples are often insufficient for reliable miRNA profiling experiments, even when using sensitive techniques such as real-time RT-PCR. This is where the miScript PreAMP PCR Kit enters.
Using 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. This breakthrough technology allows the generation of high quality, reproducible miRNA expression profiling results from limited RNA samples.
For miScript PreAMP PCR Kit, click here
Important: miScript PreAMP miRNome Primer Mixes may be provided in multiple tubes. In these cases, it is necessary to set up separate preamplification reactions and pool once the reactions are completed. For example, if 3 tubes of miScript PreAMP miRNome Primer Mix are provided, set up 3 preamplification reactions - one for each tube. After the real-time cycler run, pool the 3 reactions into one tube, dilute as described in protocol step 5, and continue with miRNA profiling as described in the protocol "Real-Time PCR for Mature miRNA Expression Profiling" in the miScript miRNA PCR Array Handbook.
Workflow for miRNA Expression Profiling using Preamplification:
Preamplification preserves expression patterns in FFPE samples using 1000-fold less input cDNA.
Preamplified cDNA and nonpreamplified cDNA from the same prep were used for miRNA profiling. Scatter plots of x-fold expression change calculations (2-ΔCT) 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 �M 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.
miScript Preamplification Unlocks miRNA Expression Profiles in Limiting Samples
Total RNA was isolated from 5 �l aliquots of normal (n=3) and colorectal cancer (n=3) serum (each isolation was from a different serum donor) using the miRNeasy Serum/Plasma Kit. cDNA was then prepared from 0.7 �l serum equivalence (SE) of each total RNA isolation in a miScript II reverse transcription reaction using HiSpec Buffer. Following reverse transcription, one-tenth of the cDNA preparation (0.07 �l SE) was used in a miScript PreAMP reaction with the Human Serum & Plasma 384HC miScript PreAMP Primer Kit. From there, the preamplified cDNA was diluted 20-fold, and 1 �l of diluted cDNA was used per well of the Human Serum & Plasma 384HC miScript miRNA PCR Array. This PCR array profiles the expression of 372 miRNAs detectable in human serum and plasma samples using the miScript PCR System. A scatter plot of 2-ΔCT values demonstrates that significant differences exist between the mature miRNA expression levels of the normal serum and colorectal cancer serum pools (Panel B, � 2-fold [red lines] used as a cutoff for significance). Those targets that exhibited differential expression include novel miRNAs as well as miRNAs whose expression has been previously shown to be regulated in colorectal cancer (hsa-miR-19b, hsa-miR-29b, hsa-miR-106b, and hsa-miR-223)1,2.
1. Arndt, G., L. Dossey, et al. (2009). "Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer." BMC Cancer 9(374).
2. Volinia, S., G. Calin, et al. (2006). "A microRNA expression signature of human solid tumors defines cancer gene targets." PNAS 103(7): 2257-61.