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We have collaborated with Biogazelle, leaders in real-time PCR, to design and experimentally validate SYBR® Green-based gene expression assays for the human and mouse transcriptome. Primer pairs for each gene were designed by prioritizing gene regions most commonly occurring in transcript variants. Every assay was wet-lab validated using iScript™ advanced cDNA synthesis kit and SsoAdvanced™ SYBR® Green supermix.
Assay Performance Standards
PrimePCR Assays: Meeting the MIQE Guidelines by Full Wet-lab Validation
Bio-Rad Laboratories has partnered with GeneGo, a Thomson Reuters business and leading provider of systems biology tools, to offer a complete pathway solution for real-time PCR-based gene expression analysis. As part of the collaboration, Thomson Reuters provided interactive pathway maps for 260 canonical pathways. Each pathway belongs to one or more general biological categories such as Cancer. The pathway maps illustrate protein interactions and regulation to provide a comprehensive picture of signaling and disease processes. The curated pathways were also used to design panels of real-time PCR assays specifically tailored to the top ranked gene targets for differential gene expression analysis. Each gene target within a pathway was assigned a score based on frequency of differential expression and popularity in scientific research both historically and currently. The resulting score were used to rank and select which assays were included on the corresponding real-time PCR pathway panel.
PrimePCR Pathway Analysis: Pathway Curation and Real-Time PCR Panel Design Strategy
Control assays and synthetic DNA templates were designed to facilitate the assessment of the key experimental factors impacting your real-time PCR results.
DNA Contamination Control AssayUse the PrimePCR DNA contamination control assay to determine if genomic DNA (gDNA) is present in a sample at a level that may affect PCR results. This assay may also be used to compare relative levels of gDNA contamination present in different samples to determine if PCR results may be affected.
Positive PCR Control AssayUse the PrimePCR positive control assay to qualitatively assess the performance of a PCR reaction associated with a single sample. This assay may also be used to compare the relative performance of PCR reactions associated with different samples.
RNA Quality AssayUse the PrimePCR RNA quality assay to determine if RNA integrity may adversely affect PCR results for a single sample. This assay may also be used to compare relative RNA integrity among different samples to determine how PCR results might be affected.
Reverse Transcription Control AssayUse the PrimePCR reverse transcription control assay to qualitatively assess the performance of the reverse transcription reaction associated with a single sample. This assay may also be used to compare the relative performance of the reverse transcription reactions associated with different samples.
Reference Gene AssaysReference genes are used in relative gene expression analysis to normalize for variation in the amount of input messenger RNA (mRNA) among samples. To ensure accurate quantitation, it is important to include one or more reference genes exhibiting constant expression levels under the experimental conditions. To streamline reference gene selection, we offer PCR primers for a set of commonly used reference genes that can be used individually, easily screened using our preplated 96-well and 384-well reference panels or added to custom-designed plates.
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PrimePCR Disease State Panels were designed by referencing the National Library of Medicine database. Disease state panels allow for the thorough investigation of differentially expressed genes within a specified pathology.
Gene targets for each disease state were ranked and compared according to the following criteria:
Frequency of gene expression changes in biomarker studies
Popularity in overall medical research
Popularity in current medical research
The resulting gene scores were used to design real-time PCR panels. For diseases with a large number of gene targets, multiple tiered plates are available. Tier 1 plates represent the highest ranked targets with subsequent tiers containing the lower ranked or less well characterized targets.