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Bio-Rad collaborated with Biogazelle, leaders in real-time PCR research, to design and experimentally validate PCR primers for gene expression assays across the human and mouse transcriptomes. All PCR primers were designed to meet stringent performance standards following the MIQE guidelines (minimum information for publication of quantitative real-time PCR experiments; Bustin et al. 2009).
Assay Performance Standards
These DNA primer pairs were designed by prioritizing the gene regions most commonly found in transcript variants. Strict design criteria were used to ensure optimal real-time PCR results for each target:
Every PCR primer pair was experimentally validated using Bio-Rad’s iScript™ advanced cDNA synthesis kit and SsoAdvanced™ SYBR® Green supermix. PrimePCR assay design and validation are fully described in the following publication.
PrimePCR Assays: Meeting the MIQE Guidelines by Full Wet-lab Validation
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 selected pathways were used to design panels of real-time PCR primers tailored for the top-ranked genes for differential gene expression analysis. Each gene target within a pathway was assigned a score based on the frequency of differential expression and its research significance. The resulting scores were used to select the assays included in 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™ PreAmp for SYBR® Green Assay: Archived Gene, Human
PrimePCR™ Template for SYBR® Green Assay: Archived Gene, Human
PrimePCR™ PreAmp for Probe Assay: Archived Gene, Human
PrimePCR™ Template for Probe Assay: Archived Gene, Human