QIAGEN Website    Quick Order    Online Seminar    Contact    My Account
Home  >  Products and Services   >  Transcriptome PCR Array  >  Complete miRNome

Complete miRNome Transcriptome PCR Arrays

 
Complete miRNome Transcriptome PCR Arrays

The Complete miRNome Transcriptome PCR Array is an innovative tool for identifying which miRNA target a gene. In each well of a Transcriptome PCR Array, is a unique "transcriptome" or cDNA sample that was synthesized from a cell sample treated with a unique miRNA mimic. Therefore, the user can screen 894 different miRNA treatments with simple qPCR reactions. The real-time PCR assay can be for any gene. Only mRNAs that can be converted to cDNA via random hexamers and oligo-dT primed reverse transcription will be tested. A change in expression of the tested gene in a specific sample reveals that the corresponding miRNA that is a regulator of the tested gene. The ten arrays are a collection of cDNAs derived from HeLa cells treated with miRNA mimics testing 894 miRNAs. Each of these arrays may be ordered individually.

 

Array Description How It Works Research Example Manuals & Data Analysis
 

Each miRNA Transcriptome PCR Array contains 90 experimental cDNA samples and 6 control samples (except for Plate 10 that tests 84 miRNAs tested). Each experimental cDNA sample is derived from HeLa cells treated with a different miRNA mimics. View the Gene Table to see the transcription factor knockdowns tested with this Transcriptome PCR Array.

Gene Table for Plate 1   Gene Table for Plate 2   Gene Table for Plate 3   Gene Table for Plate 4  Gene Table for Plate 5

Gene Table for Plate 6   Gene Table for Plate 7   Gene Table for Plate 8   Gene Table for Plate 9  Gene Table for Plate 10

Note: Each of the above arrays may be ordered individually.

 

Array Description How It Works Research Example Manuals & Data Analysis
 

Overview of Transcriptome PCR Array Protocol.

 

The Transcriptome PCR Array is comprised of a single PCR plate (chosen based on user's machine type) and data analysis software.

The user of a Transcriptome PCR Array must supply a qPCR assay that is specific for their gene-of-interest. The user may use either a SYBRŪ green or Probe-based assay with the Transcriptome PCR Arrays. To perform the assay the user must add qPCR Master mix and the assay specific for their gene to every well in the Transcriptome PCR Array. The user then runs the Transcriptome PCR Array through a cycling program specific for the selected gene-specific assay. The Ct values obtained from the instrument are then imported into the data analysis software so the user can identify regulators of the slected gene's expression.

The Excel-based data analysis software for Transcriptome PCR Arrays performs the ΔΔCt based fold-change calculations from uploaded raw threshold cycle data from the gene-specific real time-PCR assay. The spreadsheet delivers results in a tabular format and assists in hit selection.

Back to Top
 

Array Description How It Works Research Example Manuals & Data Analysis
 

Introduction
MicroRNAs are ~22 nt endogenous RNAs that function to fine-tune the expression of a target gene. Most mammalian miRNAs are found to decrease target mRNA levels resulting in decreased protein production [1]. It has been theorized that most mammalian genes are targets of miRNAs [2]. Currently, the only method to screen which miRNAs may target a gene of interest is through prediction algorithms. Different prediction algorithms use slightly different rules which can lead to substantially different results. Even after using prediction algorithms to determine which miRNAs target a gene of interest, these miRNAs must be tested experimentally to verify that they are real interactions. For a gene that may have tens or hundreds of predicted miRNAs, this can be an expensive and daunting task. In addition, prediction algorithms may miss true miRNA/target RNA interactions due to the rules that they are restricted to. miRNA Transcriptome PCR Array provides comprehensive experimental methods to screen for miRNAs that target a gene of interest.
To demonstrate the power of miRNA-FIND in discovering novel miRNA/mRNA interactions, we screened for miRNAs that target ZEB2. ZEB2 is a transcription factor that has been implicated in the epithelial-mesenchymal transition (EMT).

Material and Methods
In this study we used "Cancer miRNA SureFind Transcriptome PCR Array" to identify potential miRNAs that target ZEB2. SYBR Green qPCR assay was performed to quantify the expression of ZEB2 (gene of interest) and GAPDH (house-keeping control). Fold change in ZEB2 gene expression as a result of each specific miRNA mimic treatment relative to negative mimic control were calculated and normalized to GAPDH. Fold changes were converted to log2 and subjected to MAD analysis for positive hits selection.

Figure 1: miRNAs involved in regulation of ZEB2 gene expression. Cancer miRNA SureFind Transcriptome PCR Array was used to run SYBR Green-based qPCR assays for ZEB2 and GAPDH. ZEB2 gene expression level is expressed as Log2 fold change based on Ct calculation using GAPDH as house-keeping gene and non-target miRNA mimic treated sample well (VTC) as negative control.

Results
Our results show that miR-200c, let-7g, miR-122, miR-142-5p, and miR-29b negatively regulate ZEB2 gene expression (Fig. 1). Also, miR-34c-5p caused an increase in expression, possibly identifying it as a positive regulator of ZEB2 mRNA expression (Fig. 1). Interestingly, miR-200c has been shown to inhibit EMT through direct targeting of ZEB2 [3]. While miR-200c has been previously identified to target ZEB2, let-7g, miR-122, miR-142-5p, miR-29b and miR-34c-5p are novel regulators that are not identified by any prediction algorithm (data not included).

Conclusions
miRNA Transcriptome PCR Array identified six miRNAs that regulate ZEB2 expression and five of these (let-7g, miR-122, miR-142-5p, miR-29b and miR-34c-5p) were not predicted by bioinformatics (data not included).

References
1. Guo H, Ingolia NT, Weissman JS, Bartel DP. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature. 2010 Aug 12;466(7308):835-40.
2. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009 Jan;19(1):92-105.
3. Park SM, Gaur AB, Lengyel E, Peter ME. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev. 2008 Apr 1;22(7):894-907.

 

Back to Top

 

Array Description How It Works Research Example Manuals & Data Analysis
 

Back to Top