Research Journal of Microbiology1816-4935xxxx-xxxxScience International10.3923/jm.2015.329.335Listeria innocua]]>HeTing LiLinfang FengJia YuHaiqing 72015107Currently, little is known about a protocol in establishing the quantitative Real-Time PCR (qRT-PCR) system in gram-positive bacteria. Also, it is obscure whether the expression level of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) in Listeria innocua is influenced by the pH. This study focused on providing a protocol for analyzing Cas9 gene expression in L. innocua at two pH conditions. The 37°C overnight culture was diluted 1:500 and grown in fresh Todd Hewitt Yeast extraction broth (THY) for 3 h. The collected pellet was washed two times in pH = 7.5 medium and was cultured for 4 h by using pH = 7.5 and pH = 6.0 medium, respectively. The culture was treated with RNA protection reagent for 5 min and the obtained pellet was suspended with mutanolysin for 30 min. Total RNA was extracted by using a kit and was incubated with DNase I, which was removed by adding phenol-chloroform-isoamyl alcohol thereafter. The purified RNA was precipitated via glycogen-ethanol treatment and was synthesized into cDNA. The 2‾ΔΔCt method was used to analyze the expression level of Cas9 gene with 16S rRNA gene as a reference. The gel electrophoresis showed that the quantity of 23S rRNA was about two-fold of 16S rRNA. The relative Cas9 expression levels of L. innocua in neutral and acidic medium were 1.08±0.49 and 1.66±0.54, respectively (p>0.05). Totally, the qRT-PCR system for analyzing Cas9 gene expression in L. innocua was successfully developed. The Cas9 expression level of L. innocua in acidic medium was similar to that in neutral medium.]]>Chiang-Ni, C., P.X. Zheng, P.J. Tsai, W.J. Chuang, Y.S. Lin, C.C. Liu and J.J. Wu,2012611622Deltcheva, E., K. Chylinski, C.M. Sharma, K. Gonzales and Y. Chao et al.,2011trans-encoded small RNA and host factor RNase III.]]>471602607Han, C., D. Zhao, C. Wu, Z. Ning and J. Yang et al.,2006116164Liu, T., W. Xu and Y. Zhang,200040224227Luo, Y.L., X.P. Huang, X.M. Wu, S.T. Ding and Y.P. Ma et al.,2008Streptomyces globisporus fermentation fluid.]]>20527528Luo, C., Z.Q. Wang, F.Q. Gong and D.S. Shi,200929233237Pang, X., D. Zhou and R. Yang,200313034Sampson, T.R. and D.S. Weiss,20132013Sampson, T.R., S.D. Saroj, A.C. Llewellyn, Y.L. Tzeng and D.S. Weiss,2013497254257Shelburne III, S.A., R.J. Olsen, N. Makthal, N.G. Brown and P. Sahasrabhojane et al.,2011Streptococcus pyogenes.]]>8214811495Shu, X.L., Y.D. Wu and S.Q. Shang,200810732736Sun, Y.X., H.M. Sun, R.L. Guo, X.H. Zhang and J.Q. Yuan,200523293296Xu, D.S., X.F. Wu and P.Q. Cheng,2007Listeia monocytogenes.]]>17861863Xu, S. and X. Xu,2013Bacillus subtilis.]]>443641Yang, R.D.,2006Pages: 435Pages: 435Zhao, J., Z.C. Liu, L. Dai and M.J. Liu,2009Ziziphus jujuba mill.]]>10111117Zheng, K., Q.S. Chen and X. Li,2007Apostichopus japonicus.]]>188487Zou, X.L., L.C. Liu and L.X. Luo,20132919481954