Rnase inhibitor plus

Total RNA was isolated by hot phenol extraction and purified using RNeasy (QIAGEN). Splinted ligation of RNA was performed as described previously (Wang et al. 2013 (link)). Briefly, an RNA anchor was ligated to the free hydroxyl of a decapped rps23 mRNA facilitated by a gene-specific DNA splint. After removal of the splint by the DNase I (Roche), rps23 gene-specific RT primers were used for reverse transcription to synthesize cDNA using SuperScript III (Invitrogen) and then PCR amplified using a primer complementary to the RNA anchor and an rps23 specific reverse primer. For tobacco acid pyrophosphatase (TAP) treatment, 10 µg total RNA was decapped by treatment with 20 U of RNase Inhibitor Plus (Promega) and 5 U tobacco acid pyrophosphatase (Epicentre Biosystems) in the supplied 1× TAP buffer for 30 min at 37°C before splinted ligation of RNA.

Ten micrograms of total RNA, 20 pmol of DNA splint and 30 pmol of RNA anchor were mixed and incubated at 70°C, 60°C, 42°C and 25°C, for 5 minutes at each step. The 20 U T4 DNA ligase (Invitrogen), T4 DNA ligase buffer and 20 U RNase Inhibitor Plus (Promega) were then added and ligation allowed to proceed overnight at 15°C. Ligase was omitted for control samples. Reactions were then treated with DNase using the Qiagen DNAse kit and protocols described therein. RNA was extracted using phenol/chloroform, precipitated with 1 ml 100% EtOH and 0.3M sodium acetate for 2 h at −20°C and washed once with 70% EtOH. RNA was then resuspended in 13.3 μl RNase‐free water and used for RT‐qPCR.