Myiq2 machine

To determine homozygosity of CBA-Edn1 mice, a quantitative real time PCR assay was performed in which a region corresponding to part of the fifth exon of Edn1 was amplified using the following PCR primers: 5′-GACCATCTGTGTGGCTTCTAC-3′; 5′-GGAACACCTCAGCCTTTCTT-3′. Primer function was verified in a standard PCR reaction, in which they correctly amplified a 92 bp fragment. Primers used to detect the Ramp3 gene (the internal reference gene) have been previously described (Sakurai et al., 2008 (link)). Briefly, genomic DNA was extracted as described above for genotyping. Amplification was performed using the QuantiTect SYBR Green PCR kit (Qiagen). Each PCR reaction contained 80 ng of genomic DNA and 6.25 pmol of each primer. Reactions were carried out on the MyiQ2 machine (BioRad) using the following conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 1 min. Results were analyzed by the ΔΔCt method, in which the 2^−ΔΔCt (fold change) was calculated by subtracting the ΔCt (Ct for Edn1- Ct for Ramp3) of mutant embryos (homozygous and heterozygous) from the ΔCt of wild type embryos.

One E10.5 CBA-Edn1;Wnt1-Cre embryo from the 1398 and 1416 lines and one E10.5 control embryo were harvested and stored in RNAlater (Qiagen). Total RNA was extracted from whole embryos using the RNeasy Micro Kit (Qiagen). cDNA was prepared from total RNA using the QuantiTect cDNA Synthesis Kit (Qiagen). Real-time quantitative PCR was performed using 5 ng of cDNA and the QuantiTect SYBR Green PCR Kit (Qiagen) and QuantiTect Primer Assays for Edn1 and Actb (β-actin; Qiagen). RT-PCR and data analysis was performed using a MyiQ2 machine (BioRad). Data analysis was performed using the ΔΔCt method as described previously (Barron et al., 2011 (link)).