Expression analysis and tissue distribution for PTCHD1, PTCHD1AS1 and PTCHD1AS2 was performed by RT-PCR, with a multiple tissue panel of first strand cDNA. The housekeeping gene G3PDH was used as a control. Origene human adult brain tissue panel was used to check the expression of PTCHD mRNA in different regions of the brain. qRT-PCR was performed with TaqMan Gene Expression assay Hs00288486, and samples were pre-normalized to GAPDH expression. Northern blot analysis was performed with a six tissue mRNA blot (BioChain). The BioChain FastHyb solution was used to hybridize the probe according to manufacturer’s instructions. RNA in situ hybridization was performed on paraffin sections and whole-mounted fetal mouse and adult mouse brain using a 411 bp (chrX:152,008,934-152,009,344, UCSC Mouse July, 2007 (38)) digoxigenin-labeled mouse antisense probe (and sense probe as negative control), using standard methods. To examine cellular localization of PTCHD1 protein, full-length human fetal brain PTCHD1 cDNA was PCR amplified and cloned into the pcDNA3.1/CT-GFP-TOPO expression vector (Invitrogen). After confirming the correct sequence and orientation of the insert, we transiently transfected COS-7 and SK-N-SH cells with 2 μg of
