Figure 5

qSL-RT-PCR assay exploring the 5' cap structure of mRNAs involved in endonucleolytic cleavages in Brachypodium. (a) Schematic diagram of the qSL-RT-PCR. Solid line: mRNA; Solid rectangle: ORF (open reading frame). The mRNAs containing a 5' cap cannot be ligated to Anchor RNA, but uncapped RNAs, with a 5' monophosphate, can be ligated to the Anchor RNA 3' hydroxyl mediated by complementary DNA Splint oligonucleotides. The ligated RNA is converted to cDNA by reverse transcription with a reverse gene-specific primer. The resulting cDNA is then detected by PCR using gene-specific primer and Anchor primer (a forward primer that anneals to the anchor region). (b) Total RNA were treated with (+) or without (-) Tobacco Acid Pyrophosphatase (TAP), an enzyme removing the cap structure from capped mRNA, then annealed with anchor RNA and complementary DNA Splint oligonucleotides. The ligated RNA was converted to cDNA by reverse transcription and the resulting cDNA was detected by PCR, amplified for 30 cycles (+TAP) and 37 cycles (-TAP), respectively. The mRNAs treated with TAP were used as positive control. The ubiquitin10 (UBQ10) cDNA was used as a control to normalize the amount of cDNA in samples, and 22 cycles were run for UBQ10 amplification.