Real-time Quantitative PCR (qPCR) has become a sensitive, accurate and straight forward method for detecting DNA/RNA since Higuchi et al. pioneered this technique in the early 1990’s. It is now widely used in many biomedical research laboratories.
There are two types of DNA detection chemistry that are used in qPCR: specific and non-specific. Specific DNA sequence detection can distinguish between the sequence of interest, primer dimers and non-specific amplification. In contrast, non-specific chemistry detects all double stranded DNA produced during the reaction. The standard method for non-specific detection uses an intercalating dye that fluoresces once bound to dsDNA, the most common being SYBR Green I (excitation, 497nm emission, 520nm). Unlike specific detection systems, non-specific methods do not require unique probes, therefore they are less expensive (synthesis of custom probes can be expensive). In addition, the products detected by SYBR Green I can be quantified using a dissociation curve at the end of the reaction. Therefore, SYBR Green I is quite versatile because the same dye can be used to detect any amplified product.
For the last five months, I have been using the Eurogentec’s qPCR Master Mix for SYBR Green I Kit. This kit contains 2X reaction Master mix which has been mixed with all the necessary components for a qPCR reaction from start to finish. A typical reaction includes 5.0 ng RNA or cDNA, 0.1uM primer and 1x qPCR Master mix in 20 ul total volume and standard cycling conditions (ABI) are applied. The whole process takes around 4 hours (1.5 hours for sample preparation and 2.5 hours for completion of the reaction).Using the Eurogentec qPCR Master Mix for SYBR Green I Kit, I have analyzed more than 20 gene expression levels from cultured cell lines, primary cells or different tissues of mice and rats. These genes are involved in the regulation of cell survival, proliferation, and differentiation. The results among different batches of samples have been sensitive and reproducible.
In addition, the Eurogentec qPCR Master Mix for SYBR Green I Kit can be useful in detecting gene mutations. The dsDNA melting point can be observed as a decrease in fluorescence (520 nm) as SYBR Green I dissociates. Thus, different size and/or sequence products will melt at different temperatures yielding distinct peaks for the first negative derivative of fluorescence vs temperature. For a fully optimized PCR reaction, the melting peak profile should contain only one single peak, which represents the specific product expected from the primer pair. Therefore, amplicons that differ by a single nucleotide will melt at slightly different temperatures and can be distinguished by their melting peaks. In this way it is possible to distinguish homozygotes (single peak) from heterozygotes (two peaks).
The Eurogentec qPCR Master Mix for SYBR Green I Kit is a very useful tool to study gene expression and mutation. The sensitivity, reproducibility and convenience of this product make it an asset in any laboratory. I am happy with this product and would recommend it to other scientists.
Hongyun She
Senior Scientist
Department of Pathology
University of Southern California