Figure 2

Mapping of of hnRNPD transcription start site in oral cancer cells. (A) RLM-RACE assay was used for mapping the transcription initiation site of human hnRNPD gene. Total RNA isolated from SCC-4 cells after treatment with Calf Intestinal Phosphatase (CIP) was decapped with tobacco acid pyrophosphatase. Then GeneRacer RNA adaptor was ligated to the 5ʹend of mRNA using T4 RNA Ligase. The adaptor ligated mRNA was reverse transcribed and subjected to primary and secondary PCR using nested adaptor and gene specific primers. 18S RNA was reverse transcribed and used as positive control and PCR reaction without template served as negative control. The PCR products were reolved on 1.2% agarose gel. (B) The prominent 250 bp band obtained after secondry PCR was subjected to double strand DNA sequencing after clonning into TA cloning vector pCR4. The first nucleotide after the RNA adaptor sequence was considered the transcription start site and has been marked as + 1.