Introduction
Tobacco use is one of the major threats to human health which kills nearly 6 million people each year with an estimated 8 million deaths in the year 2030. More than 2,500 chemicals are found in tobacco and its smoke, among which terpenoids and alkaloids are the major groups [1]. Though there is clinical uncertainty about the carcinogenic property of nicotine, evidences have proved nicotine as the main psychoactive agent responsible for the development of tobacco dependence [2, 3]. Nicotine diffuses readily into brain tissue where it binds to nicotinic acetylcholine receptors reinforcing addiction. It is estimated that 3-5 mg/day is the threshold level that readily establishes and sustains addiction among smokers [4]. The LD50 of …show more content…
First strand cDNA was synthesized with DNase I treated RNA and antisense primers for the reference gene (β-ATPase), target genes (PMT1/5, PMT2/4, PMT3 and HCT) (supplementary table 1). In a total reaction volume of 30µl, RNA (3µg) dNTP’s (10mM) antisense primers (5picomoles) and DTT (5mM) were added and denatured at 65oC for 5minutes. Then, the samples were plunged into ice. To this reaction mix, 50 Units of reverse transcriptase (MuMLV) and reverse transcription assay buffer (5X) was added and incubated at 30ºC for 5 minutes. First strand cDNA was synthesized at 42ºC for 60minutes. Thereafter, the abundance of the PMT members and HCT was studied using gene specific sense primers by qPCR (Light Cycler LC480, Roche Diagnostics). Sense and antisense primers were designed using IDT. Sense primers specific for genes and antisense primers for a conserved region of the PMT genes. Quantitative PCR was performed in a 20µl reaction using Light Cycler 480 SYBR Green I master mix as per the manufacturer’s protocol. Previously, individual primer pairs were validated for PCR efficiency using the slope obtained in qPCR. All experiments were carried out in
Tobacco use is one of the major threats to human health which kills nearly 6 million people each year with an estimated 8 million deaths in the year 2030. More than 2,500 chemicals are found in tobacco and its smoke, among which terpenoids and alkaloids are the major groups [1]. Though there is clinical uncertainty about the carcinogenic property of nicotine, evidences have proved nicotine as the main psychoactive agent responsible for the development of tobacco dependence [2, 3]. Nicotine diffuses readily into brain tissue where it binds to nicotinic acetylcholine receptors reinforcing addiction. It is estimated that 3-5 mg/day is the threshold level that readily establishes and sustains addiction among smokers [4]. The LD50 of …show more content…
First strand cDNA was synthesized with DNase I treated RNA and antisense primers for the reference gene (β-ATPase), target genes (PMT1/5, PMT2/4, PMT3 and HCT) (supplementary table 1). In a total reaction volume of 30µl, RNA (3µg) dNTP’s (10mM) antisense primers (5picomoles) and DTT (5mM) were added and denatured at 65oC for 5minutes. Then, the samples were plunged into ice. To this reaction mix, 50 Units of reverse transcriptase (MuMLV) and reverse transcription assay buffer (5X) was added and incubated at 30ºC for 5 minutes. First strand cDNA was synthesized at 42ºC for 60minutes. Thereafter, the abundance of the PMT members and HCT was studied using gene specific sense primers by qPCR (Light Cycler LC480, Roche Diagnostics). Sense and antisense primers were designed using IDT. Sense primers specific for genes and antisense primers for a conserved region of the PMT genes. Quantitative PCR was performed in a 20µl reaction using Light Cycler 480 SYBR Green I master mix as per the manufacturer’s protocol. Previously, individual primer pairs were validated for PCR efficiency using the slope obtained in qPCR. All experiments were carried out in