Monitoring mRNA Half-Life in Arabidopsis Using Droplet Digital PCR

Abstract

mRNA decay is an important process in post-transcriptional regulation; in addition, it plays a crucial role in plant development and response to stress. The development of new tools to quantify mRNA decay intermediates is thus important to better characterize the dynamic of mRNA decay in various conditions. Here, we applied droplet digital PCR (ddPCR), a recent and precise PCR technology, to determine mRNA half-life in Arabidopsis seedlings. We demonstrated that ddPCR can correctly assess mRNA half-life from a wide variety of transcripts in a reproducible manner. We also demonstrated that thanks to multiplexing mRNA, the half-life of multiple transcripts can be followed in the same reaction. As ddPCR allows precise quantification, we proposed that this approach is highly suitable when a low amount of RNA is available; for the detection of many targets or for the analysis of lowly expressed transcripts.

Genome-wide analysis of mRNA decay in Arabidopsis shoot and root reveals the importance of co-translational mRNA decay in the general mRNA turnover

“…We selected a transformant line expressing transgene levels similar to that of endogenous XRN4 and transgenic XRN4ΔCTRD (Supplemental Figure 13B). We next measured half-lives of candidate transcripts, through ddPCR-monitoring of RNA levels following cordycepin treatment (30), in wild-type, xrn4 lof, XRN41′CTRD and XRN41′CTD(R 118 A/R 119 A) backgrounds (Figure 7 and Supplemental Figure 14). First, to confirm that the XRN41′CTRD allele retains normal and similar catalytic activities both in root and shoot, we selected three transcripts amongst the list of those identified as only targeted by the XRN4 cytosolic decay in both organs (subgroup 4.2, Figure 5A-B).…”

Genome-wide analysis of mRNA decay in Arabidopsis shoot and root reveals the importance of co-translational mRNA decay in the general mRNA turnover

“…We selected a transformant line expressing transgene levels similar to that of endogenous XRN4 and transgenic XRN4ΔCTRD ( Supplementary Figure S12B ). We next measured half-lives of candidate transcripts, through ddPCR-monitoring of RNA levels following cordycepin treatment ( 33 ), in wild-type, xrn4 , XRN4ΔCTRD and XRN4ΔCTD(R 118 A/R 119 A) backgrounds (Figure 8 and Supplementary Figure S13 ). First, to confirm that the XRN4ΔCTRD allele retains normal and similar catalytic activities both in root and shoot, we selected three transcripts amongst the list of those identified as only targeted by the XRN4 cytosolic decay in both organs (subgroup 4.2, Figure 6A – B ).…”

“…cDNAs were then diluted 50-fold prior to ddPCR analysis. ddPCR was performed as described previously ( 33 ). Luciferase quantification in each sample was used for normalization.…”