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Cancer Transcription Factor Knockdown Transcriptome PCR Array

 
Cancer Transcription Factor Knockdown Transcriptome PCR Array

The Cancer Transcription Factor Knockdown Transcriptome PCR Array is an innovative tool for identifying which transcription factors regulate the expression of any 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 siRNA. Therefore, the user will screen 90 different siRNA treatments with a single qPCR reaction. 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 transcription factor is a regulator of that gene. This array is a collection of cDNAs derived from MCF-7 cells treated with siRNAs that knockdown the expression of 90 cancer-associated transcription factors.

 

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Cancer Transcription Factor Knockdown Transcriptome PCR Array contains 90 experimental cDNA samples and 6 control samples. Each experimental cDNA sample is derived from MCF-7 cells treated with a different siRNAs. View the Gene Table to see the transcription factor knockdowns tested with this Transcriptome PCR Array.

Gene Table

 

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 SYBRŽ 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.

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Identification of transcription repressors that regulate the expression of HMOX1 gene with SureFIND Transcriptome PCR Array

Introduction
Heme oxygenase-1 (HMOX1) is an inducible enzyme that catalyzes oxidative degradation of heme to form biliverdin, carbon monoxide and free iron. HMOX1 expression is elevated in advanced stage breast-, gastric- and prostate- cancers and shown to have protumoral function. HMOX1 response also plays a role in drug resistance during chemotherapy (1). Although function of HMOX1 gene has been intensively studied, the molecular mechanism behind gene expression regulation of HMOX1, especially the repression mechanism that normally active when no stimulants available is important for understanding the tumor development and its drug resistance. Transcription Factor Knockdown Transcriptome PCR Array provides comprehensive experimental methods to screen for transcriptional regulators for a gene of interest.

Materials and methods
In this study, we employed Cancer Transcription Factor Knockdown Transcriptome PCR Array to simultaneously analyze the transcription regulators for HMOX1 gene expression. SYBR green qPCR Assay was performed to quantify the expression of HMOX1 (gene of interest) and RPL13A (house keeping gene). Fold change in HMOX1 gene expression as a result of each transcription regulator specific siRNA treatment relative to negative siRNA control were calculated and normalized to RPL13A. Fold changes were converted to log2 and subjected to MAD analysis for positive hits selection.

Figure 1. Screening of HMOX1 gene expression repressors with SureFIND. MCF7 cell based SureFIND Transcriptome PCR Array (Cancer Transcription Factor Knockdown) was used with its 90 targets gene indicated. RT˛ qPCR Primer Assays (SYBRŽ Green-based) for HMOX1 and RPL13A was performed. HMOX1 gene expression level are expressed as Log2 fold changes based on Ct calculation using RPL13A as house-keeping gene and non-target siRNA treated sample well (VTC) as negative control.

Results
Our results show that BACH1, MYBL2 and YY1 negatively regulate HMOX1 gene expression. Knock down of BACH1, MYBL2 and YY1, showed about 12-fold, 8-fold and 10-fold increase in the level of HMOX1 mRNA compared to the negative control, which was from cells treated with non-targeting siRNA, respectively (Figure1). Interestingly, BACH1 is well known repressor for HMOX1 expression (2), whereas MYBL2 and YY1 are novel regulators.

Conclusions
Transcription Factor Knockdown Transcriptome PCR Array identified three transcription factors that repress HMOX1 gene expression and two of these (MYBL2 and YY1) were novel finding.

References
1. Jin Song, et al. (2009) Suppression of Annexin A11 in Ovarian Cancer: Implications in Chemoresistance. Neoplasia. 11(6): 605.
2. Jiying Sun, et al. (2002) Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene. The EMBO Journal. 21(19). 5216

 

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Array Description How It Works Research Example Manuals & Data Analysis
 

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