ABgene QPCR Overview

Introduction

The Quantitative Polymerase Chain Reaction (QPCR) is one of the most powerful and sensitive gene analysis techniques available and is used extensively in industrial, academic and diagnostic labs. QPCR is routinely used for a broad range of applications including gene expression analysis, genotyping, SNP analysis, viral load testing, pathogen detection, drug target validation and for measuring RNA interference.

Historically, quantitative analysis of nucleic acid templates was tackled using Northern and Southern blot hybridization or conventional end-point PCR. Using these techniques, a sample of unknown concentration was estimated by comparing the intensity of the amplified band on a gel or blot to standards of a known concentration.

However, in conventional PCR the results are collected at one time-point, usually after the reaction is complete, making it impossible to determine the starting concentration of the template nucleic acid. Consequently, these approaches gave only ‘semi-quantitative’ results.

In QPCR, accumulation of PCR product is measured and reported throughout the amplification reaction, instead of at just one time-point. This is why QPCR is often referred to as real-time PCR analysis. When an amplification reaction is in the log phase, the quantity of PCR product is directly proportional to the amount of input nucleic acid. QPCR is truly ‘quantitative’ because it allows measurements to be taken during this phase of the reaction.

A QPCR reaction contains a fluorescent reporter molecule to monitor the accumulation of PCR product. As the quantity of target amplicon increases, so does the amount of fluorescence emitted from the fluorophore. QPCR reactions containing a higher concentration of template take less time to accumulative a threshold concentration of PCR product, while those containing less template take longer. The Ct value, which is the number of PCR cycles that elapse before the threshold is reached, is a measure of the input nucleic acid (see Figure 1). By comparing the results of samples of unknown concentration with a series of standards, the amount of template DNA in an ‘unknown’ reaction can be accurately determined.

Figure 1. After 30 cycles the amount of fluorescence detected for each dilution is very similar due to limiting reagents. Traditional endpoint analysis after 30-40 cycles would not enable accurate quantification of starting material.

In QPCR, the amplification reaction is performed using a thermal cycling instrument but the fluorescent signal is measured using a fluorescence detection system incorporated in the QPCR instrument.    

Absolute and Relative Quantitation

The quantity of target nucleic acid can be measured using either absolute or relative quantitation. Absolute quantitation measures the overall amount of target nucleic acid whereas relative quantitation measures the ratio between the target nucleic acid and a reference, usually a housekeeping gene. By comparing the target to a reference, relative quantitation generates a normalized value that can be used to compare the level of gene expression in different samples.   

Absolute Quantitation

This strategy relies on a series of external standards to determine the absolute amount of target nucleic acid. These standards usually contain sequences that are the same, or very similar to, the sequence of the target gene. However, the sequences of the primer binding sites must be identical to the target sequence to ensure the amplification efficiency of the standard and target molecule are the same. There is a linear relationship between the Ct value and the log of the standard for a dilution series. Consequently, if the dilution series has been prepared correctly, plotting Ct against log standard gives a straight line. This is the standard curve used to measure the quantity of target in ‘unknown’ samples. Comparing the Ct of unknown samples against the standard curve allows calculation of the initial amount of target used in real-time RT-PCR.

Relative Quantitation

This is an alternative quantitation strategy that examines the ratio of the target nucleic acid to a reference molecule within the same sample. The Ct value of the target is divided by the Ct value of a reference to give a ‘normalized’ value. The normalized value can then be compared against other samples to determine differential gene expression in different samples. This quantitation strategy is most commonly used for analysis of gene expression.

Typically, reference molecules are constitutively expressed which means they are often referred to as housekeeping genes. The challenge for the researcher is to find a good housekeeping gene, one that is neither up- nor down-regulated significantly under experimental conditions. It’s very difficult to find a reference gene that meets this criterion. Consequently, it’s common for researchers to normalize their target gene against multiple housekeeping genes to minimize variation.

Primer and Amplicon Design

Efficient and specific amplification is absolutely essential for reproducible QPCR analysis. The sequence of both the primers and probes has a very significant influence on QPCR amplification. Therefore, the target and primer sequences must be chosen carefully to maximize the chances of QPCR success.

The following is a list of criteria that the amplicon sequence should meet:

• Shorter amplicons are amplified with more efficiency. Consequently, the amplicon should be between 75 and 200 bp in length. The amplicon should be at least 75 bp to distinguish it from any primer dimers that might form.
• Secondary structure in the amplicon should be avoided because this will hinder amplification. Programs such as mfold can model the amplicon to see if there is significant secondary structure at the reaction annealing temperature.
• The GC content of the amplicon should be between 50 and 60%. A higher proportion of GC encourages secondary structure.
• The template should have no more than 4 repeats of a single base because this encourages non-specific amplification.

For primer design, the following guidelines should be adhered to:

• Primers should be designed outside of any secondary structure in the target sequence to promote amplification efficiency.
• Primers should be no more than 50-60% GC-rich. A higher GC content increases the likelihood of secondary structure and primer-primer interaction.
• The melting temperature (Tm) should be between 50°C and 65°C. Annealing temperatures outside this range cause poor amplification efficiency and / or specificity.
• Repeats of Gs or Cs longer than 3 bases should be avoided because this encourages non-specificity.
• The sequence of the forward and reverse primers should not share 3’ complimentarity at their 3’ termini as this promotes primer-dimer formation.   The Basic Local Alignment Search Tool (BLAST) should be used to confirm that there is only one homologous sequence in the target molecule.