Real-time PCR (qPCR) is the preferred method for DNA and cDNA quantification because of its high sensitivity, reproducibility, and wide dynamic range .
KAPA SYBR® FAST qPCR Kits are recommended for gene expression studies using the sequence non-specific intercalating dye SYBR® Green I.
KAPA PROBE FAST qPCR Kits are recommended for gene expression studies using sequence specific TaqMan® probes, FRET probes, and molecular beacons.
Gene Expression – Housekeeping Genes
Despite the precision of qPCR, conclusions drawn from gene expression experiments are often misleading due to differences in amplification efficiency between the gene of interest and the housekeeping gene(s). The pitfalls in performing relative quantification with non-uniform amplification efficiencies were demonstrated by Pfaffl et al. . For example, a difference in PCR efficiency (∆E) of 10% between target gene and reference gene falsely calculated differences in expression ratio of 7.2 % in the case of ETarget < ERef and 1083 % in the case of ETarget > ERef after 25 performed cycles . Differences in amplification efficiency can result from either sub-optimal primer design and/or difficult amplicon sequence (e.g. high GC content).
KAPA SYBR® FAST qPCR Kits contain the first DNA polymerase engineered specifically for SYBR® Green I-based qPCR through a process of molecular evolution. The KAPA SYBR® DNA Polymerase exhibits improved speed, processivity and robustness resulting in consistently high amplification efficiencies required for accurate relative quantification. To demonstrate the high performance of the KAPA SYBR® FAST qPCR Kit for gene expression analysis, the reaction efficiencies obtained for ten commonly used housekeeping genes in the human breast cancer cell line, MCF-7, were compared. The KAPA SYBR® FAST qPCR Kit achieved consistently high amplification efficiencies (95 - 104%) across all ten genes, despite differences in amplicon length and GC content.
Gene Expression – Relative Quantification
Relative quantification using qPCR measures the changes in steady-state mRNA levels of a gene across multiple samples normalized to a reference gene(s). In theory, the expression levels of the reference gene (often referred to as the housekeeping gene) should remain stable in the tissues or cells under investigation or in response to the experimental treatment. In practice, there is considerable evidence that housekeeping gene expression varies significantly [3 – 8]. Despite this fact, many gene expression studies still make use of internal control gene(s) without validation of the presumed stability of expression. The geNorm algorithm developed by Vandosompele et al. (2002)  enables rapid and accurate determination of the most stable reference genes from a set of tested genes in a given cDNA sample and is considered the gold standard for determining the most suitable set and number of housekeeping genes to use for accurate relative quantification.
One challenge when using multiple housekeeping genes for relative quantification is the requirement for high amplification efficiencies (95 - 105%) across all genes, regardless of amplicon length, complexity or GC content. KAPA SYBR® FAST qPCR Kits contain the first DNA polymerase engineered specifically for SYBR® Green I-based qPCR through a process of molecular evolution. The KAPA SYBR® DNA Polymerase exhibits improved speed, processivity and robustness, resulting in consistently high amplification efficiencies required for accurate relative quantification using a panel of diverse housekeeping genes (see Application Note Gene Expression I: Housekeeping Genes above).
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