(A) Schematic illustration of the ePAT approach. In the first step of the assay, a DNA oligo is annealed to adenylated RNA via an oligo-(dT) stretch. The addition of Klenow polymerase and dNTPs to the reaction results in templated extension of the 3′ terminus of the poly(A) tract to fill in the recessed end. The second step of the assay is performed at 55°C to ensure that the primer used for extension can only remain annealed to end-extended molecules and not internal poly(A) tracts through oligo-(T) alone. PCR amplification of cDNA using a gene-specific primer (primer 1) and a universal reverse primer (primer 2) results in a range of amplicon sizes that reflects the position of the gene-specific primer relative to the poly(A) site and the length distribution of poly(A)-tails in the sample. (B) Serially diluted yeast total RNA, as indicated, was spiked into a fixed concentration (800 ng) of total HeLa RNA and analyzed by ePAT. The 200-ng sample was also used to prepare a TVN-PAT reaction. PCR amplicons from TVN-PAT and ePAT reactions were resolved on 2% high-resolution agarose gels and imaged. The TVN-PAT reaction represents the size of the amplicon with a fixed (A12)-tail. The signal intensity, but not the poly(A)-tail length distribution, of the two yeast genes GAL10 and APQ12 decreases with decreasing total RNA concentration. The human GAPDH adenylation state and concentration remains constant. (C) The signal from the GAL10 and APQ12 transcripts was quantitated by peak-finding and densitometry. The normalized signal (to 100% y-axis) was plotted against concentration (x-axis) and expressed in log2 to show the linear relationship between the signal and the RNA concentration.
