5′ rapid amplification of cDNA ends (5′RACE)

Total RNA was isolated from TR cells, which are a necortical neuroblast clonal cell line of mouse origin transformed by large T antigen and vras (Chun and Jaenisch, 1996). Total RNA extraction was via the guanidine isothiocyanate method (Chomczynski and Sacchi, 1987). TR is a necortical neuroblast clonal cell line of mouse origin transformed by large T antigen and vras (Chun and Jaenisch, 1996). These cells stably express telencephalon-specific gene BF-1 and a gene enriched in the neocortical ventricular zone, vzg-1. An antisense Lpar1 oligodeoxynucleotide (5′-CCGGTTGCTCATTCGT GTATGGAGCTG-3′) corresponding to the center region of exon 3 was synthesized (Contos and Chun, 1998). The synthesis of the first cDNA strand and subsequent amplification of 5′ cDNA end was carried out as detailed in the BD SMART RACE cDNA Amplification kit manual. Total RNA was reverse-transcribed using a modified lock-docking oligonucleotide (dT) primer and BD SMART II oligonucleotide at 42°C for 1.5 h to obtain the first cDNA strand. 5′RACE was performed by incubating the antisense Lpar1 antisense primer with the first cDNA strand, using the following PCR conditions. After an initial denaturation of one cycle at 94°C for 2 min, the mixture was amplified at 94°C for 45 s, at 68°C for 45 s, and at 72°C for 3 min for 30 cycles. The resulting products were cloned into a sequencing vector, TOP cloner TA, and sequenced to determine the transcription start site.