Figure 6

LM-PAT assay measuring the mRNA poly(A) tail length for genes involved in endonucleolytic cleavages in Brachypodium. (a) Schematic diagram of the qSL-RT-PCR. Solid line: mRNA; Solid rectangle: ORF (open reading frame). The mRNA is saturated with p(dT)_12-18 (small rectangles filled with oblique lines)_, which allows the ligation of adjacent ones. Then, oligo(dT) anchor is ligated to the 3'- most oligo(dT) (small empty rectangle). The synthesis of cDNA from the RNA sample is carried out using the oligo(dT) anchor. PCR is performed with oligo(dT) anchor primer and gene-specific primer. (b) Total RNA was extracted from Brachypodium seedlings with (C) and without cold treatment (W). Bradi1g56580, an endogenous-cleavage-unrelated type II gene, was used as positive control. PCR products from the LM-PAT assay were analyzed on 2.2% Metaphore agarose gels.