Universal probe library based assays

Tissues were harvested from tips of palatal shelves from E14 and E15 mouse embryos, placed into 200µl of RLT (RNeasy mini kit, Qiagen), and RNAs were isolated by using RNeasy columns (Qiagen). cDNAs were synthesized by using Omniscript reverse transcriptase (Qiagen) according to the manufacturer’s protocols. Real time quantitative PCR experiments were done either by using Universal Probe library-based assays (Roche Applied Science) or by using TaqMan assay reagents (Applied Biosystems) (see Table II). 30µl assays were quantified using Applied Biosystems ABI7300 PCR and ViiA7 detection systems and software. Data were normalized to β-actin mRNA levels using the 2^−ΔΔCt method.

Most experiments were carried out using Universal Probe Library-based assays (Roche Applied Science) with gene-specific primer sequences generated by the manufacturer’s online algorithm and TaqMan Universal PCR master mix (Applied Biosystems) (see Supplemental Table 3 for sequences). Some used Taqman Assay reagents (see Supplemental Table 3). 30µl assays were quantified using Applied Biosystems ABI7300 PCR and ViiA7 detection systems and software. All Ct values were checked by eye. Actb levels were used to normalize other expression levels. cDNA was diluted to avoid Cts lower than 18. At least three separate control (Cre negative or conditional heterozygote) and three separate mutant samples were assessed for each genotype/condition, where possible from the same litter, or at least stage-matched.