Our goal is to provide low cost qPCR primer sets to validate NGS or microarray data and quantitate gene expression using real time qPCR assays

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Overview - Quantitative "Real Time" PCR or qPCR

Quantitative Real-Time PCR (qPCR), often simply called qPCR, has revolutionized our ability to precisely measure nucleic acid concentrations. This powerful technique uses qPCR primers to provide a highly accurate and reproducible method for quantifying DNA or RNA, making it an indispensable tool in modern molecular biology.

Unlike traditional PCR, real-time PCR instruments monitor the accumulation of a PCR product at each step of the reaction, providing "real-time" data on amplification kinetics. This innovation significantly streamlined the process, moving beyond the laborious post-PCR analysis required by older methods like gel electrophoresis or HPLC.

The widespread adoption of qPCR has been driven by its enhanced ease, accuracy, and reproducibility. Its core principle relies on detecting fluorescence generated during amplification, with SYBR green being a popular and reliable method due to its simplicity.

Key Applications of qPCR The precision and speed of quantitative real-time PCR have opened doors to a diverse range of critical applications across research and diagnostics, including:

Validation of Gene Expression Data: Essential for confirming findings from microarray analysis, RNA sequencing (RNA-Seq), and other genomics techniques. DNA Copy Number Measurement: Accurately determining the number of gene copies in a sample. Detection and Quantitation of Pathogens: Highly effective for identifying and quantifying viral particles, bacteria, and other microorganisms. Mutation and SNP Analysis: Identifying genetic variations with high sensitivity. miRNA Expression Analysis: Quantifying microRNA levels for various biological studies. How qPCR Works: The Fundamentals At its core, a real-time PCR protocol shares similarities with standard PCR, requiring a clean template, well-designed qPCR primers, and optimized reaction conditions. The key distinction lies in the incorporation of a fluorescent reporter – either an intercalating agent like SYBR green or fluorescently labeled probes/primers – to detect PCR product accumulation.

As the qPCR reaction proceeds exponentially, fluorescence increases. The threshold cycle (Ct value) is a critical metric, defined as the point where fluorescence significantly rises above the baseline. Crucially, the Ct value is inversely proportional and linearly related to the initial amount of target nucleic acid. By comparing Ct values across samples, researchers can accurately calculate target concentrations and assess qPCR efficiency, making real-time PCR an unparalleled technique for quantitative analysis.

PCR products may be quantitated by generating a standard curve or quantitated relative to a control gene. Real time PCR quantitation based on a standard curve may utilize plasmid DNA or other forms of DNA in which the absolute concentration of each standard is known. One must be sure, however, that the efficiency of PCR is the same for the standards as that of the "unknown" samples. Performing PCR from purified targets can in some cases be more efficient than that observed with complex nucleic acid mixtures. The relative quantitation method is somewhat simpler as it requires the measurement of housekeeping or control genes to normalize expression of the target gene. However, the selection of appropriate control genes can cause problems as they may not necessarily be equally expressed across all unknown samples. This may be circumvented by normalizing measurements to a set of housekeeping genes in order to avoid this variability problem.

In addition to robust qPCR primers, a critical aspect of performing real time PCR is to begin with a template that is of high purity. This can be challenging when working with some biological samples. Fortunately, a number of commercial products have been developed to facilitate the isolation of nucleic acids in high purity. Removing contaminating phenol and unwanted DNA are steps to consider. For gene expression studies, reverse transcription must be carried out with high purity reagents and in multiple replicates as this step can introduce variability in template replication. Reverse transcription may be done prior to real time PCR or may be incorporated within the amplification program.