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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.
Show Reference Gene AssaysView Reference Gene Assay Panels
Plectin is a prominent member of an important family of structurally and in part functionally related proteins termed plakins or cytolinkers that are capable of interlinking different elements of the cytoskeleton. Plakins with their multi-domain structure and enormous size not only play crucial roles in maintaining cell and tissue integrity and orchestrating dynamic changes in cytoarchitecture and cell shape but also serve as scaffolding platforms for the assembly positioning and regulation of signaling complexes (reviewed in PMID: 9701547 11854008 and 17499243). Plectin is expressed as several protein isoforms in a wide range of cell types and tissues from a single gene located on chromosome 8 in humans (PMID: 8633055 8698233). Until 2010 this locus was named plectin 1 (symbol PLEC1 in human; Plec1 in mouse and rat) and the gene product had been referred to as "hemidesmosomal protein 1" or "plectin 1 intermediate filament binding 500kDa". These names were superseded by plectin. The plectin gene locus in mouse on chromosome 15 has been analyzed in detail (PMID: 10556294 14559777) revealing a genomic exon-intron organization with well over 40 exons spanning over 62 kb and an unusual 5' transcript complexity of plectin isoforms. Eleven exons (1-1j) have been identified that alternatively splice directly into a common exon 2 which is the first exon to encode plectin's highly conserved actin binding domain (ABD). Three additional exons (-1 0a and 0) splice into an alternative first coding exon (1c) and two additional exons (2alpha and 3alpha) are optionally spliced within the exons encoding the acting binding domain (exons 2-8). Analysis of the human locus has identified eight of the eleven alternative 5' exons found in mouse and rat (PMID: 14672974); exons 1i 1j and 1h have not been confirmed in human. Furthermore isoforms lacking the central rod domain encoded by exon 31 have been detected in mouse (PMID:10556294) rat (PMID: 9177781) and human (PMID: 11441066 10780662 20052759). The short alternative amino-terminal sequences encoded by the different first exons direct the targeting of the various isoforms to distinct subcellular locations (PMID: 14559777). As the expression of specific plectin isoforms was found to be dependent on cell type (tissue) and stage of development (PMID: 10556294 12542521 17389230) it appears that each cell type (tissue) contains a unique set (proportion and composition) of plectin isoforms as if custom-made for specific requirements of the particular cells. Concordantly individual isoforms were found to carry out distinct and specific functions (PMID: 14559777 12542521 18541706). In 1996 a number of groups reported that patients suffering from epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) lacked plectin expression in skin and muscle tissues due to defects in the plectin gene (PMID: 8698233 8941634 8636409 8894687 8696340). Two other subtypes of plectin-related EBS have been described: EBS-pyloric atresia (PA) and EBS-Ogna. For reviews of plectin-related diseases see PMID: 15810881 19945614. Mutations in the plectin gene related to human diseases should be named based on the position in NM_000445 (variant 1 isoform 1c) unless the mutation is located within one of the other alternative first exons in which case the position in the respective Reference Sequence should be used. [provided by RefSeq Aug 2011]
PrimePCR™ PreAmp for SYBR® Green Assay: PLEC, Human
PrimePCR™ Template for SYBR® Green Assay: PLEC, Human
PrimePCR™ PreAmp for Probe Assay: PLEC, Human
PrimePCR™ Template for Probe Assay: PLEC, Human
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Aurum™ Total RNA Mini KitThe Aurum total RNA mini kit produces high-quality DNA-free total RNA from a wide range of starting materials including cultured cells, bacteria, and yeast, as well as animal and plant tissues.
iScript™ Advanced cDNA Synthesis Kit for RT-qPCRThe iScript advanced cDNA synthesis kit for RT-qPCR is a simple, fast, and sensitive first-strand cDNA synthesis kit for gene expression analysis by reverse-transcription qPCR.
PrimePCR™ Assays and PanelsExperimentally validated real-time PCR assays are available as individual assays, preplated pathway- and disease-specific panels, or custom- configured plates.
SsoAdvanced™ Universal Real-Time PCR SupermixThe SsoAdvanced™ universal real-time PCR supermixes employ our patented Sso7d fusion enzyme technology for higher processivity, increased PCR inhibitor tolerance, and robust performance with difficult qPCR targets. Available for probe- and dye-based assays in ROX- dependent or ROX-independent real-time PCR detection systems.
Real-Time PCR Detection SystemsReal-time PCR amplification systems combine thermal cyclers with optical reaction modules for singleplex and multiplex detection of fluorophores. All systems feature thermal gradient functionality.
Amplification SoftwareSoftware for Bio-Rad thermal cyclers supports multi-instrument control and collection and analysis of real-time data. Data analysis tools include ΔCq and ΔΔCq analyses and scatter plot, volcano plot, heat map, and clustergram outputs.
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View the products required to complete your real-time PCR experiments.
Pricing for PrimePCR real-time PCR assays is based on assay type, the number of reactions, and plate configuration for 96- and 384-well plates.
Discount applies to bulk orders of the same catalog number and is taken off the list price.
Bulk discounts are shown below; download the pricing flyer for full details.
Real-Time PCR Assays and Controls
5 or more assays
Pathway and Collection Panels
20 or more plates
Custom PlatesCustom 96-well & 384-well plates with 1–96 unique genes
Custom 384-well plates with 97 or more unique genes
30 or more plates
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