A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons

Abstract

We describe a rapid, high-throughput method to scan for new RNA editing sites. This method is adapted from high-resolution melting (HRM) analysis of amplicons, a technique used in clinical research to detect mutations in genomes. The assay was validated by the discovery of six new editing sites in different chloroplast transcripts of Arabidopsis thaliana. A screen of a collection of mutants uncovered a mutant defective for editing of one of the newly discovered sites. We successfully adapted the technique to quantify editing of partially edited sites in different individuals or different tissues. This new method will be easily applicable to RNA from any organism and should greatly accelerate the study of the role of RNA editing in physiological processes as diverse as plant development or human health.

Figure 1.

Procedures to scan transcripts for new editing sites (a) and quantify editing (b) by high-resolution melting analysis of amplicons. A real-time PCR is done in the presence of a fluorescent double-stranded DNA-specific dye. At the end of the amplification, amplicons are denatured, renatured and then a high-resolution melting is performed. The decrease in fluorescence is precisely monitored as the temperature increases causing the denaturation of the DNA molecules and the release of the fluorescent dye. The presence of less thermostable heteroduplexes in a sample alters the shape of the melting curves. (a) To scan transcripts, primers spanning the transcripts are designed to give PCR products with a maximum size of 600 pb. For each primer pairs, the shape of the melting curve of a control genomic DNA (cont) is compared to the melting curve of a mix of genomic DNA and cDNA (exp: experiment). If there is no editing site, both control and experiment (e.g. exp1 shown here, amplicon chloro96) exhibit the same curve shape and are called as a single group. If there is an editing site within the amplicon, the curve of the experiment will be different from the control [as shown in exp2; rps14 (37161) within the chloro72 amplicon] and two groups of curves will be called. The differences in the curves are easily visualized by plotting the difference in signal between control and experiment (shown at the bottom of the panel). (b) To quantify editing in a sample, primers are designed to generate small amplicons flanking the editing site. Editing standards are produced by cloning amplicons from genomic DNA for the unedited standard (0%) and from an edited cDNA sample (100%) and by mixing unedited and edited plasmids with increments depending on the required degree of quantification accuracy. The derivative (−dF/dT) of the fluorescence signal is plotted to show the melting peak. The shape of the melting peak of a cDNA sample is compared to the shape of the melting peak of the standards, giving an estimate of the extent of editing in the sample (here around 50%).

Figure 2.

HRM screening of Arabidopsis chloroplast transcripts. Six new sites were identified by a high-throughput screen of all chloroplast transcripts. The duplicate fluorescence difference melting curves of controls (genomic DNA) and experiments (mix of gDNA and cDNA) are shown for the six new sites.

Figure 3.

HRM screening of the Arabidopsis clb19 mutant. The clb19 mutant is altered in the editing of two sites: rpoA (genome position 78691) and clpP (genome position 69942). In both cases, the melting curve corresponding to clb19 has the same shape as the unedited genomic control, indicating no detectable editing. A site from rpoC1 is shown as a control where clb19 and wild-type samples have similar curves.

Figure 4.

Comparative quantification of editing by HRM and PPE. (a) Standards used for the quantification: melting peaks (−dF/dT) of amplicons containing the sequence surrounding the editing site in ndhG (genome position 118858) with an increasing ratio of T as compared to C at the editing site (0–100%). (b) Determination of the extent of editing of ndhG (118858) in WT rosette and roots. The percentage of edited molecules is determined by comparison of the shape of the melting peak with the standards. It is given as a range between the values for the two standard curves on either side of the sample curve. (c) Poisoned primer extension assay to quantify editing of ndhG (118858). An RT-PCR is done with primers surrounding the editing site which then serves as a template for the extension reaction of a fluorescent oligonucleotide binding near the editing site. The extension is stopped by the incorporation of ddGTP at the editing site if the molecule is not edited or at the next C if it is edited. The extent of editing is determined by the ratios of fluorescence intensity between the two extension products in the gel.