Figure 2. sPAT accurately measures poly(A) tail lengths of artificial mRNAs and requires a small RNA input. (A) The presence of a DNA splint enhances detection levels of artificial mRNAs with defined poly(A) lengths of 105 (top), 60 (middle), and 15 (bottom) adenosines. PAT assays were performed with the given mRNA amounts in the absence or presence of a DNA splint. A single primer pair targeting the 3′UTR and the RNA anchor was sufficient to measure accurately the length of the poly(A) tail; i.e., a second nested PCR was not required. (B) sPAT is accurate and sensitive. It detects accurate poly(A) tail lengths of in vitro transcribed artificial mRNAs in the absence (lanes 2–10) or presence (lanes 11–19) of 1 µg of Xenopus oocyte total RNA. Target mRNA amounts and PCR primer combination as in (A). (C) sPAT is more sensitive than ePAT and recapitulates poly(A) tail lengths without internal priming. PCR products from 22 (top) and 28 (bottom) PCR cycles are displayed. The arrowhead points at an truncated poly(A) tail product that occurred due to an internal priming event of the anchor primer, lanes 8 and 14. All products were verified by Sanger sequencing.