Another gene analyzed is the calcium-activated potassium ion channel gene (KCNN3) involved in neural excitability and in migraine susceptibility.
The gene analyzed (MDN1) is, however, 14 kb long.
The gel was visualized under ultraviolet light to identify the specific band for each gene analyzed.
A third gene analyzed, Atp9b, localized distally from Sall3 but ubiquitously expressed, presented only open chromatin modifications.
was used to prepare cDNA from 50 ng of total RNA for each gene analyzed as previously described [35].
The last gene analyzed was Arc and, once again, we found significant differences between means of experimental groups [F (4, 25, 19.01), p<0.0001].
Power SYBR green, TaqMan master mix, and Taqman gene expression assays were purchased from Applied Biosystems (Foster City, CA) and used to determine cDNA abundance for each gene analyzed.
In contrast with results from the fibroblast cell line, where a clear mono or biallelic expression pattern was observed for each gene analyzed, many placental samples showed intermediate allelic ratios (Figure 2D) that were quantified with the PeakPicker software [26].
Real time PCR was performed using Taqman universal PCR master mix (Applied Biosystems, UK) and primers and a fluorescent TaqMan probe specific for each gene analyzed or HPRT (used for normalization).
Expression levels of target genes in the ashh2 mutant were calculated relative to wt levels with normalization to ACTIN2, using the formula RE = EtargetΔCPtarget (wt- ashh2)]/Eactin[ ΔCPactin (wt – ashh2)], where target represents the target gene analyzed.
Student's t-test (two-tailed, assuming unequal variance) was used to determine statistical significance (p-values) for differences in the relative expression values between tissues for each gene analyzed.
