Figure 2

NKX2-1-AS1 follows tissue-specific patterns of expression similar to NKX2-1 in human cells. (A) Expression of NKX2-1-AS1 and NKX2-1 determined by RT-PCR in lung cell lines. The NKX2-1-AS1 PCR fragments were sequenced to confirm the identity of the sequence. (B) Relative expression patterns of NKX2-1-AS1 and NKX2-1 in tissues and cell lines, including normal human adult lung and thyroid and H441 and H661 cell lines, as determined by qPCR (n = 3; *p < 0.05; **p < 0.01). (C) 5′-RACE analysis of NKX2-1-AS1 in human thyroid identified multiple transcription initiation sites within the 500 bp 5′ of the transcription initiation site reported in Ensembl. Thyroid total RNA was used as it has higher levels of NKX2-1-AS1 expression than the lung. The sensitivity of the method did not allow us to analyze lung RNA. Thirty clones, generated in three independent experiments, were sequenced. The black bar indicates the region recently identified in a genome wide analysis of accurate lncRNA transcription initiation sites¹³. (D) Time course analysis of NKX2-1-AS1 and NKX2-1 transcript stability in H441 cells by qPCR after inhibition of transcription by actinomycin D treatment (n = 3; *p < 0.05).