Gel express: a novel frugal method quantifies gene relative expression in conventional RT-PCR

Mohamed Hazman

doi:10.1186/s43088-022-00194-3

Abstract

BackgroundReal-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression. Nevertheless, compared with conventional PCR, the real-time PCR system is extremely expensive and not affordable for limited or mid-budget research laboratories. Here, a novel, doable and low-cost recipe (referred to as gel express) is developed to quantify gene expression using conventional RT-PCR assay. The novelty of the gel express method is based on replacing crossing point (CP) values with integrated density (IntDen) values of PCR amplicon bands in real-time PCR regular mathematical formulas.ResultsIn this work, gene expression profiles of two different rice stress-marker genes (OsCYP94C2a and OsLOX8) were quantified in response to mechanical wounding at different time points (0, 30, 60, and 150 min). In the gel express method, the free software ImageJ was employed to measure integrated density (IntDen) values of PCR amplicon bands in agarose gel images. IntDen values were then used instead of crossing point (CP) values according to the following modified formula: [EIntDen(ref)/EIntDen(target)]sample ÷ [EIntDen(ref)/EIntDen(target)]control. Gene relative expression profiles (dynamic expression pattern) quantified by gel express method in both genes were highly comparable with real-time RT-PCR. R2 values were 0.9976 and 0.9975 in OsCYP94C2a and OsLOX, respectively. PCR amplification efficiency (E) for all studied genes could be calculated depending on IntDen values through experimentally designed calibration curves. PCR amplification efficiencies with all studied genes obtained by gel express were all in the accepted range. For better-visualized PCR amplicons thus detectable biological effects between treatments, the number of PCR cycles applied in gel express method (IntCyc) was experimentally estimated to be 29 cycles.ConclusionsGel express is a novel, cost-effective and feasible recipe for quantifying gene relative expression in conventional RT-PCR. The expression pattern quantified by gel express is highly comparable and fits the expression data revealed by the used real-time PCR system.

Highlights

Real-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression
The integrated density of each PCR amplicon band visualized in raw agarose gel images was measured using ImageJ and plotted against each corresponding cycle (Fig. 1a)
The PCR amplicon band was initially detectable to eyes after 22 cycles in actin, 24 cycles in OsCYP94C2a, and 28 cycles in OsLOX8 (Fig. 1b)

Summary

Introduction

Real-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression. Compared with conventional PCR, the real-time PCR system is extremely expensive and not affordable for limited or mid-budget research laboratories. A novel, doable and low-cost recipe (referred to as gel express) is developed to quantify gene expression using conventional RT-PCR assay. The novelty of the gel express method is based on replacing crossing point (CP) values with integrated density (IntDen) values of PCR amplicon bands in real-time PCR regular mathematical formulas. There are main four methods for quantifying specific gene expression: Northern blotting and in situ. It is required to confirm candidate gene expression to validate RNA-seq data and reduce errors of wide-range sequencing platforms [10]. The real-time PCR system is extremely expensive and not accessible for a wide range of research laboratories. There are some additional essential running costs such as kits and special disposable (PCR tubes/plates with special lids) which, if not fairly provided, the system could be paused

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Conclusion