Gene:  COL5A1, Human

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:

• Target regions without SNPs
• PCR primer pairs annealing across intron/exon junctions when possible
• No secondary structure in primer annealing sites
• Maximum number of transcript isoforms detected
• PCR primers compatible with standard assay conditions

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

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 Assay
Use 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 Assay
Use 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 Assay
Use 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 Assay
Use 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 Assays
Reference 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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