For each 1 μg of DNAse I-treated RNA, 50 ng of random

hexamers (Invitrogen) and 10 nM of each deoxynucleoside triphosphate (dNTPs, Bioline) were added and the mixture incubated at 65°C for 5 min, then immediately cooled on ice. To this, 4 μl of 5 x first strand reaction buffer (Invitrogen) and dithiothreitol (Invitrogen) to a final concentration of 0.1 M were added and the mixture incubated at 25°C for 2 min, then 1 μl (200 U) of Superscript II reverse transcriptase (RT) (Invitrogen) was added and this website the reaction incubated for 10 min. A negative control (no RT) was also included, with 1 μl of RNase-free water substituted for the Superscript II reverse transcriptase. The reverse transcription reactions were incubated at 42°C

for 50 min. The reaction was stopped by incubation at 70°C for 15 min and the total volume made up to 600 μl with nuclease-free water and aliquots stored at −20°C. Each qRT-PCR reaction was conducted in a 20 μl volume and contained 5 μl template cDNA, 10 μl of 2 x Platinum SYBR Green qPCR Supermix containing Rox Dye (Invitrogen) and 100 nM each of the PRTF and PRTR primers (Table 1). Reactions Selleck PLX4032 were run using a Stratagene MX3000P. Each assay included test cDNA, the no-RT control reaction previously described and a no template control, to which only water was added. The cycling conditions were an initial incubation for 2 min at 50°C, followed by 5 min at 95°C, then 40 cycles of 95°C for 30 s and 60°C for 30 s. Reactions were carried out in triplicate for each sample. Relative quantification of phoA transcription was normalised against transcription from the glyceraldehyde 3-phosphate dehydrogenase gene Amobarbital (GAPDH, GeneID: 1090024) using the HLF and HMR primers (Table 1) and the relative level of expression calculated

using the delta-delta Ct method [41]. Detection of alkaline phosphatase activity in cultured cells Mycoplasma transformants were grown in 10 ml MB supplemented with gentamicin at 16 μg/ml until an approximate pH of 7.2 was reached, then pelleted by centrifugation at 20,000 x g for 20 min at 4°C. The cells were resuspended and washed twice in ice-cold 0.05 M Tris, pH 8.0 (T buffer) and again centrifuged and washed as before. The cells were finally resuspended in T buffer with 1% Triton X-100 (ICN) added and incubated for 15 min at 4°C. The total protein concentration of the cell lysate was determined in triplicate using a BCA kit (Pierce) following the manufacturer’s instructions. To determine the AP activity of each transformant in triplicate, 10 μl of the cell lysate was added to reaction buffer (1 M Tris, pH 8.0, 1 mM MgCl2) to which 50 μl of 2 mM disodium p-nitrophenyl phosphate (pNPP, Calbiochem) in reaction buffer was added and the mixture incubated at 37°C for 30 min. The reaction was terminated by addition of 100 μl 2 M NaOH and the absorbance read at 410 nm using a spectrophotometer (Labsystems Multiskan MS).