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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 are designed to aid in assessing the key experimental factors that may be impacting your real-time PCR results.
DNA Contamination Control AssayThe PrimePCR™ DNA contamination control assay is designed to determine if genomic DNA (gDNA) is present in a sample at a level that may affect PCR results. The 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 AssayThe PrimePCR positive control assay is designed to qualitatively assess the performance of a PCR reaction associated with a single sample. The assay may also be used to compare the relative performance of the PCR reactions associated with different samples.
RNA Quality AssayThe PrimePCR RNA quality assay is designed to determine if RNA integrity may adversely affect PCR results of a single sample. The assay may also be used to compare the relative RNA integrity between different samples in order to determine if PCR results may be affected.
Reverse Transcription Control AssayThe PrimePCR reverse transcription control assay is designed to qualitatively assess the performance of the reverse transcription reaction associated with a single sample. The 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) between samples. For accurate quantitation, it is important to use one or more reference genes that are shown not to change expression as a result of the experimental treatment or condition. To aid in this, we have suggested a set of commonly used reference genes that can be used individually, easily screened using our pre-plated 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.
PrimePCR pathway plates allow for gene expression profiling or investigation of differentially expressed genes within a specific pathway. Gene targets for each pathway were ranked and compared according to frequency of gene expression changes in transcriptome studies and popularity in overall and current research. For pathways 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.