PrimePCR Pathways

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PrimePCR Assays - Pathway Panels
 

Why PrimePCR?

PrimePCR™ Lookup Tool

Use the PrimePCR lookup tool to find assays and panels for your genes of interest.

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Assay Design and Validation

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

Sensitivity Accurate detection of 20 copies
Specificity Amplicon sequence validated with next generation sequencing (NGS); Minimal primer dimer formation and genomic DNA cross reactivity.
Amplification Efficiency 90–110%
Linear Dynamic Range Minimum of six orders of magnitude; Detection of a synthetic template standard curve from 20 to 20 million copies.
R2 >0.99

PrimePCR Assays: Meeting the MIQE Guidelines by Full Wet-lab Validation

Pathway Curation

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

Controls and References
Controls

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 Assay
The 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 Assay
The 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 Assay
The 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 Assay
The 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 Assays
Reference 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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Reference genes


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