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. www.selleckchem.com/products/mln-4924.html 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 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 TGF-beta inhibitor 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 Staurosporine ic50 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.