Table 2.

Total RNA was extracted from postmortem DLPFC tissues of eight adult normal controls (from CBDB/NIMH Brain Collection) and was used to synthesize cDNA as described previously (Lipska et al., 2006)a

NameSequence
NKCC1 Ex1 forward primer5′-GGTCGGGCAGCTATGGAGCCGCGG-3′
NKCC1 Ex27 reverse primer5′-TGGAGCAACTTTCCTGTGTGCTTTCA-3′
NKCC1 Ex1 forward primer5′-GGTCGGGCAGCTATGGAGCCGCGG-3′
NKCC1 Int4 reverse primer5′-GGGTCACAGTGATTTGGATCCTCTGG-3′
NKCC1 Ex1 forward primer5′-GGTCGGGCAGCTATGGAGCCGCGG-3′
NKCC1 Int2 reverse primer5′-TCCCTGCTCAGCCTTCTTGATTCTGA-3′
  • aIn humans, at least two different NKCC1 transcripts (NM_001046 and NM_001046.2) have been reported in the NCBI RefSeq database. PCR primers used for this study were designed on the basis of these existing NKCC1 transcripts' sequences (Table 2). Portions of the NKCC1 gene were amplified with each combination of primers using Platinum Taq DNA polymerase (Invitrogen). The PCR conditions were 94°C for 5 min, 35 cycles of 94°C for 30 s, 58°C-62°C for 30 s, 3 min, 72°C for 30 s, and 72°C for 7 min. The sequences of amplified products were analyzed by direct sequencing to evaluate the presence of novel transcripts. The exon structure of each product was determined by aligning the amplified sequence to the human chromosome 5 genomic contig sequence of the human NKCC1 gene (NT_034772.6). Subsequently, the same analysis was conducted using postmortem human DLPFC tissues of eight adult normal controls and frontal cortical tissues of eight fetal prefrontal cortical samples from CBDB/NIMH Brain Collection (Tan et al., 2008). Based on the sequence information of 5′- and 3′-ends obtained from RACE, we designed primers for amplification of full-length transcripts (Table 2).