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5 ��mol/L forward and reverse primers, 4 mmol/L MgCl2, and 2�� SensiMix SYBR Green PCR Master Mix (Bioline, Sydney, NSW, Australia). The cDNA was amplified by 1 cycle at 95��C for 15 minutes followed by 36 cycles of 95��C for 15 seconds (denaturing), 55��C for 20 seconds (annealing), and 72��C for 25 seconds (extension) using a C1000TM Thermal Cycler and CFX96TM Real-Time System (Bio-Rad Laboratories). Melting point analysis indicated that only a single product was produced in each reaction and confirmed by preliminary runs with gel electrophoresis which also established that only one product of the predicted size was produced. Control runs of all PCR experiments contained non-template controls and negative reverse transcriptase www.selleckchem.com/products/crenolanib-cp-868596.html controls consisting of RNA samples where reverse transcriptase was not PTPRJ added (such that no cDNA was produced) to test for DNA contamination. Statistical Methods Each quantitative PCR sample was analyzed in duplicate and efficiency of reactions was determined using linear regression of the Log (fluorescence) per cycle number data with the LinRegPCR program40 and ranged from 2.008 �� 0.006 (��-actin) to 1.877 �� 0.008 (5-HT3 receptor C subunit [HTR3C]). Expression data was calculated relative to ��-actin and GAPDH and expressed as the following ratio: Ratio = (Efficiencyreference)Ct reference/(Efficiencysample)Ct sample, where Ct is the crossing point threshold of the sample for the amplified genes. Relative expression data was further analyzed after log transformation using one-way ANOVA followed by Tukey��s multiple comparisons test. One-way ANOVA and Tukey��s multiple comparisons were used to analyze the number of patients expressing genes detected using end point PCR. The number of observations used to derive mean values is expressed by n and arithmetic mean values are given as mean �� SEM. Results Distribution of 5-HT3 Receptor Subunits in the Human Intestine The distribution of 5-HT3 receptor subunits was examined using quantitative reverse transcript PCR on samples obtained from throughout the length of the colon and the ileum area of the small intestine. In all regions, expression was examined in both the mucosal and muscular tissue layers and reported relative to expression of ��-actin. GAPDH was used as a second housekeeping gene for comparison with a previous selleckchem study where the relative expression of the 5-HT3 receptors in the sigmoid colon was reported.22 Control RNA samples incubated without reverse transcriptase and then amplified with GAPDH primers were also undertaken to demonstrate that there was no DNA contamination of the RNA extractions (data not shown). GAPDH levels of expression were consistently and significantly higher than those of the 5-HT3 receptor subunits or RIC3 in both ileum and colon (P
