Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis

TLDR

Real‑time PCR is increasingly used for mRNA quantification, enabling rapid gene expression analysis from low template amounts, yet all data‑analysis methods share common principles and are broadly classified as absolute or relative. The study evaluates absolute and relative quantitative PCR analysis approaches to assess nocturnal c‑fos expression in wild‑type and retinally degenerate mice. The authors compare multiple absolute and relative methods and apply a simple algorithm that calculates amplification efficiency from each sample’s amplification profile, automating the process to streamline analysis. They confirm nocturnal c‑fos expression originates from the photoreceptor layer, showing a ~5‑fold reduction in rod‑cone‑deficient mice, and demonstrate that accounting for sample‑specific amplification efficiency yields comparable results without standard curves, enabling automated, kinetics‑based analysis.

Abstract

Real‐time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. Despite a wide range of approaches, the same principles underlie all data analysis, with standard approaches broadly classified as either absolute or relative. In this study we use a variety of absolute and relative approaches of data analysis to investigate nocturnal c‐fos expression in wild‐type and retinally degenerate mice. In addition, we apply a simple algorithm to calculate the amplification efficiency of every sample from its amplification profile. We confirm that nocturnal c‐fos expression in the rodent eye originates from the photoreceptor layer, with around a 5‐fold reduction in nocturnal c‐fos expression in mice lacking rods and cones. Furthermore, we illustrate that differences in the results obtained from absolute and relative approaches are underpinned by differences in the calculated PCR efficiency. By calculating the amplification efficiency from the samples under analysis, comparable results may be obtained without the need for standard curves. We have automated this method to provide a means of streamlining the real‐time PCR process, enabling analysis of experimental samples based upon their own reaction kinetics rather than those of artificial standards.