Sharbati et al. BMC Genomics 2012, 13:23 http://www.biomedcentral.com/1471-2164/13/23 1 μM representing 32.3 nM of each DNA-oligonucleo- tide. The reaction was performed in 200 μl PCR tubes using the Veriti® Thermal Cycler (Life Technologies). Between 500 ng and 1 μg total RNA and 1 μl of the RT- 6-Pool was first prepared in a 5 μl volume. The mixture was incubated at 70°C for 5 min and chilled on ice. After adding 5× RT-Buffer, 1 mM dNTPs and 200 U Reverse Transcriptase, the volume was adjusted to 10 μl by add- ing nuclease-free water. The reaction was started at 37°C for 5 min followed by 42°C for 2 h. The enzyme was inac- tivated by heating at 95°C for 5 min. Afterwards the cDNA pool was cooled down for at least 1 min on ice and 90 μl of nuclease-free water was added yielding the template for qPCR arrays. RT reactions serving for com- parative analyses were always run in parallel using identi- cal amounts of total RNA. RT-qPCR arrays and conventional miR-Q Arrayed triplicate measurement of 26 miRNAs and 5 reference snRNAs was performed in 10 μl final reaction volume by means of SYBR Green detection chemistry using the SensiMix SYBR Hi-ROX Kit (Bioline GmbH) and the Step One Plus Cycler (Life Technologies). All reactions were carried out using clear MicroAmp® Fast 96-Well Reaction Plates (Life Technologies) that were sealed with adhesive films. Firstly, a master mix was pre- pared in a 945 μl volume consisting of 2× SensiMix SYBR Hi-ROX, 100 nM of each universal primer (MP-fw and MP-rev) and 100 μl of diluted RT-reaction. After dispen- sing 9 μl of the master mix in wells of a 96 well plate, 1 μl of each miRNA- or snRNA-specific reverse primer (’miRNA or snRNA’-rev) was added to respective wells of the array at a final concentration of 5 nM. The amplifica- tion was carried out via the first step at 95°C for 10 min, followed by 40 cycles with 15 s at 95°C, 20 s at 60°C and 20 s at 72°C. The fluorescence signal was acquired at 60°C. Quality control was performed by subsequent melting curve analysis by 95°C for 1 min, 60°C for 2 min, ramping from 60°C to 95°C at 2°C/min and acquiring the signal. Samples containing water instead of the specific ‘miRNA or snRNA’-rev primer served as negative controls (NC). Conventional miR-Q was performed as described pre- viously [9]. Normalisation of miRNA expression and comparative quantification Normalisation of miRNA expression was performed using a set of snRNAs: SNORD44 (NR_002750.2), SNORD47 (NR_002746.1), SNORD48 (NR_002745.1), SNORD52 (NR_002742.2) and RNU6 (NR_004394). After calculating the Cq mean of each reference snRNA, the Cq geometric mean of all reference snRNAs was used to normalise the miRNA expression values. The difference between the Cq of the miRNA of interest (Cq miRoI) and the calculated Page 10 of 11 geometric mean (Cq norm) was calculated yielding the ΔCq sample or ΔCq calibrator, respectively. The relative quantifica- tion (ΔΔCq) was performed by determining the difference between ΔCq sample and ΔCq calibrator. Fold differences were determined by calculating 2 to the power of-ΔΔCq. Additional material Additional file 1: Quality control of presented miR-Q assays. Calculated parameters (e.g. slopes, Y-intercepts and r2) for all presented miR-Q assays are given according to the MIQE guidelines. Acknowledgements and funding The authors are grateful to Barbara Kutz-Lohroff for excellent technical assistance. Furthermore we thank Dr. Pawel Janczyk for providing Salmonella serovar Typhimurium DT104. This work was supported by the Deutsche Forschungsgemeinschaft (SH 465/1-1 and SFB 852 project B4). Authors’ contributions SS conceived the study, performed experiments and analyses, wrote and edited the manuscript. JS contributed to the experiment design, data analysis and writing of the manuscript. LH and MB performed experiments. RE contributed to the writing of the manuscript. 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