Who Desires To Know How You Can Make It To The Smoothened Agonist Top Spot — различия между версиями
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One microgram total RNA from each sample was incubated with 1?��L (1?U/��L) RNase-free DNase I (Fermentas, Burlington, ON, Canada) at 37��C for 30?min and then incubated with 1?��L EDTA (25?mm) at 65��C for 10 min. The first stand cDNA was synthesized using First Strand cDNA synthesis kit with ReverTra Ace-��-reverse R428 transcriptase (TOYOBO, Kita-ku, Osaka, Japan) according to the manufacturer��s protocol. RT-qPCR was carried out with a Roche LightCycler480 Real-time PCR Detection System using SYBR Green Real-time PCR Master Mix (QPK-201) kit (TOYOBO). The primers of C-type lectin used in RT-qPCR were designed by Wei et?al. (19), and other primers were designed using Beacon Designer 7.80 software and listed in Table?1. ��-actin was used as the reference gene. The PCR contained 5?��L SYBR? Green Real-time PCR Master Mix, 0��2?��L of each primer (10?��m), 0��5?��L diluted cDNA template and filled up with 4��1?��L PCR degree water to the final volume of 10?��L. The cycling protocol was predenaturation at 95��C for 2?min followed by 40 cycles of 95��C for 15?s, 57��C for 15?s and 72��C for 20?s. Specificity of PCR products was detected by melting curve analysis and sequencing. Each sample was tested in triplicate. The relative mRNA expression of target genes to the reference gene was calculated using the 2?����Ct method (20). For inflammatory response analysis, gill was collected from infected and uninfected (as negative control) grouper at day 3 post-primary Smoothened Agonist clinical trial challenge. Samples were fixed ABT-737 solubility dmso in chilled 10% neutral buffered formalin for 24?h, followed by dehydration in a graded series of ethanol. The gill was then embedded in paraffin, cut into 5-��m sections and stained with haematoxylin and eosin following standard procedures. The photomicrographs were taken using an Olympus BX51TIF microscope. All data were analysed using spss software (version 16.0; SPSS Inc., Chicago, IL, USA). Data were expressed as mean?��?SE, the significant difference between the C.?irritans infected group and the control group at each time point was determined by Duncan��s test (P?