Gene Sequences Analysis Of Puma Exon2-4 And MRNA Expression In Basal Cell Carcinoma

Objective To analyze the mutations of puma Exon2-4 and the expression of puma mRNA in order to obtain a clue of possibly relevant to basal cell carcinoma.Methods Study on the optimized reaction conditions of three Exons of puma with high GC content. Developed a real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) for the detection of expression of puma gene mRNA.Results1. By exploring the experiment conditions of PCR, the ultimate reaction contions of three exons of puma were eatablished.(1) The reaction system of exon2 were:2×Mastermix 25μl, primers(10μmol/L) 1.25μl, template DNA 2.5μl, ddH2O 20μl, the total volume was 50μl. The amplification condition was:After 5 min at 95℃, reactions were cycled through 45s denaturation at 95℃,45s annealing at 70℃, and 45s extension at 72℃for 33 cycles; followed for 5 min.(2) The reaction system of exon3 were:primers(10μmol/L) 1.30μl, other components were same as exon2.The amplificat condition was:45s annealing at 66℃, other amplification condition were same as exon2.(3) The reaction system of exon4 were:primers(10μmol/L) 0.9μl, other components were same as exon2.The amplificat condition was:45s annealing at 59℃, other amplification condition were same as exon2.2. By using the optimized reaction conditions, the exon 2 to 4 of puma gene in 24 cases of basal cell carcinoma tissues and 7 control cases of normal skin tissues were amplificationed. puma mutations in exon 2 to 4 of amplification productions were detected by sequencing technique. The sequencing results compare to DNA bank sequence by Blast 2, none of mutation was found. 3. pMD18-T(+)/puma and pMD18-T(+)/GAPDH plasmids were successfully constructed by TA-colony. By exploring the conditions, the fluorescence quantitative RT-qPCR was developed for the study of puma gene expression level in human BCC tissue. we showed that the optimized fluorescencequantitative RT-qPCR reaction mixtures contained 2×SYBR premix 10μl; the forward and reverse primers 0.4μl each; ROX 0.4μl; cDNA 1.0μl; ddH2O7.8μl; the total reaction volume was 20μl. The RT-qPCR detecting puma was performed under the following conditions:95℃for 5 minutes; followed by 5 cycles of 95℃for 10 seconds,60℃for 5 seconds and 72℃for 10 seconds; then followed by 40 cycles of 95℃for 10 seconds,60℃for 5 seconds, 72℃for 10 seconds,78℃for 2 seconds and 72℃for 30 seconds(fluorescence detection); and 65℃for 5 minutes. The RT-qPCR detecting GAPDH was performed under the following conditions:95℃for 5 minutes; followed by 5 cycles of 95℃for 15 seconds,64℃for 5 seconds and 72℃for 10 seconds; then followed by 40 cycles of 95℃for 15 seconds,64℃for 5 seconds,72℃for 10 seconds,78℃for 2 seconds and 72℃for 30 seconds(fluorescence detection); and 65℃for 5 minutes.4. The expression levels of puma mRNA were detected by absolute quantification. The significant difference between basal cell carcinoma tissues and normal skin tissues was found(P<0.05), and the expression levels of puma mRNA in BCC group were lower than that of control group.Conclusions1. The three Exons coded sequences of puma gene are conservative and stable.2. The significant difference of between basal cell carcinoma tissues and normal skin tissues was found(P<0.05), and the relative expression levels of puma mRNA in BCC group were lower than that of control group.