Construction and validation of the mouse Refs/CLPs standard DNA. (A) Schematic representation of the mouse Refs/CLPs standard DNA used for qPCR analysis. We ligated the standard DNAs of the CLPs and the reference genes (Refs) DNA [40, 41] using EcoRI restriction sites and the resulting fragment was then cloned into the pGEM-T Easy vector. The linearized standard DNA was amplified from the plasmid DNA. To examine whether the standard DNA gave one melting temperature, we amplified the mouse Refs/CLPs standard DNA using the Ym1 (B) and Ym2 (C) primers. (D) The Ym1 and Ym2 PCR products were evaluated using 10% polyacrylamide gel analysis. (E) Multiple products were amplified from the mouse Refs/CLPs standard DNA using the Ym2 primers. Pink arrows indicate Ym2 primers. Lines indicate the putative amplified DNA products.