5 μL double-distilled water (ddH2O). The protocol followed for each qPCR was as follows: hot start
at 95°C for 10 s, followed by 45 cycles at 95°C for 5 s, 60°C for 20 s. Data were collected and analyzed using Opticon Monitor software V3.1 (BIO-RAD). To normalize the data, primer pairs were designed to amplify the gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) as housekeeping control. Based on the gene classification, 10 genes were selected for the PCR amplification and the specific primer sets that were used are listed in Table 4. STAT inhibitor The specificity of each resulting amplicon was validated with its corresponding melting curve. The relative level of expression was calculated by comparing the difference in the threshold cycle Protein Tyrosine Kinase inhibitor number of the gene of interest gene with that of the reference gene. Table 4 Primers used for real-time PCR in this study gene Sequences of primers (5′ to 3′) Amplicon size (bp) cwh TGGTAAATGCCCCATCTAGTC Selleckchem Semaxanib 137 GGCTGTAACACCAATAATTTCC hprk GAAACCCCTGTTGTCATAGTGG 126 CAATTCTCCCGATAGACGACTG
ss-1616 ACAGGGAATAAGCATCAGCG 119 ATGTAGTTACGCTCCGCCTT ysirk GCACTTTTATTGCCACGGATT 160 CAGCACCTTGTTGTCTCGGA gapdh TTGGAAGCTACAGGTTTCTTTG 98 TTACCACCAGGAGCAGTGACA ss-1955 ATCAGGTTCTAACATTGTTGCG 122 TAACGCCCCCCTCTAACAAG srt GGTCGACGAAGTGTCATTGC 123 ATACGTCAGCGTCCTCCCAC nlpa CTGCAACCTGGTCACCAAATAC 129 ACCCCGGAAAAGTTACGTATGA sdh TAGAAGTCCCTTGTGTCAGACG 134 AGATCCCACTTGGTACATAGCG ss-1298 TGGATATCGACAGCAAGGAG 156 CATAGTCGCCCAAATAGAGC trag TCGTGACTTGATGACGGCTG 167 GATAATGCCACCAGCGTTCA Colony PCR analysis To learn about gene distribution in diverse SS2 isolates with different backgrounds, colony PCR was used. The primers used
to detect the 10 IVI genes were same as the oligonucleotides for qPCR (Table 4). Single SS2 colonies were picked from THA plates, suspended in 50 μL of ddH2O and boiled for 10 min to make DNA lysates. Each was assayed using the appropriate primer sets by PCR. PCR reactions were carried out using Taq polymerase according to the manufacturer’s recommendation (TaKaRa). Acknowledgements This work was selleck compound supported by the National Basic Research Program (No. 2006CB504400) from Ministry of Science and Technology of the People’s Republic of China. We appreciate the thoughtful comments of Drs. Huochun Yao, Hongjie Fan, Yongjie Liu, Rongmei Fei, Jianhe Sun, Yaxian Yan, Jianluan Ren, and Yong Yu. We thank Miss Kaicheng Wang for kindly providing rGAPDH for this study, and Dr. Yuling Ma and Mr. Piren Chen for their assistance in sera collection. We also thank Dr. H.E. Smith for providing the SS2 T15 Strain. We are extremely grateful to Dr. Xiuguo Hua for providing SPF minipiglets. Electronic supplementary material Additional file 1: Swine convalescent sera preparation. The data provided represent the preparation of swine convalescent sera. * Time-point of antibody check. ‡ Sacrificed and serum collection.