Sharbati et al. BMC Genomics 2012, 13:23 http://www.biomedcentral.com/1471-2164/13/23 Page 9 of 11 Figure 6 Salmonella infection of human monocytes reveals a specific miRNA response. MiRNA regulation is shown in three groups using the monocytic THP-1 as a differentiation model. Fold differences between differently treated THP-1 and untreated controls are shown. Columns filled with small squares show monocytic THP-1 stimulated with viable Salmonella. Columns filled with big squares represent macrophages derived from PMA treated THP-1 and striped columns represent the third group being composed of differentiated macrophages that were infected with viable Salmonella. The bars indicate the SD of triplicate experiments each measured in triplicate. The green dashed area indicates balanced expression between samples and control. identified in differentiated as well as infected human monocytes by means of the presented method and will provide a basis for our future studies. Methods Cell culture and RNA isolation The human acute monocytic leukemia cell line THP-1 (DSMZ ACC 16) was cultured in suspension using RPMI 1640 (Biochrom AG) supplemented with 10% fetal bovine serum superior (Biochrom AG) and 10 μg/ml gentamicin (Biochrom AG) and was passaged twice weekly. THP-1 were in vitro differentiated into macrophages by adding 250 ng/ml phorbol-12-myristate-13-acetate (PMA). Differ- entiated cells were lysed after 28 h and total RNA was iso- lated using the miRVana Isolation Kit (Life Technologies). RNA integrity numbers (RIN) were determined using an Agilent 2100 Bioanalyzer and RNA Nano Chips (Agilent). All RNA isolations used in this study possessed RINs higher than 9. Infection experiments Monocytic THP-1 were seeded in 6-well plates without gentamicin at a density of 5 × 105 cells per well. After- wards, they were stimulated with viable Salmonella sero- var Typhymurium DT104 at a multiplicity of infection (MOI) of 10 in triplicate. 2 h post stimulation adherent as well as non-adherent cells were washed twice with warm PBS and lysed for total RNA isolation. Non-treated THP-1 served as controls. Macrophages derived from monocytic THP-1 were also infected with Salmonella serovar Typhymurium DT104. For this purpose, 3.5 × 105 cells per well were seeded in 6-well plates and differ- entiated as described above. Macrophages remained either non-infected or were infected with exponential- phase Salmonella serovar Typhymurium DT104 at an MOI of 10. 2 h post infection supernatants were removed and adherent cells were washed twice with warm PBS. Adherent macrophages were lysed and total RNA was isolated as described above. Oligonucleotides All miRNA specific oligonucleotides for RT as well as qPCR were designed as described earlier [9] and were synthesised and HPLC purified by Metabion AG and Eurogentec Deutschland GmbH. The miRNA sequences were taken from the miRNA database miRBase 17 [29]. Individual and multiplexed reverse transcription RT of miRNAs for single RT-qPCR was performed as described earlier [9]. For multiplexed RT, a pool of miRNA- as well as reference snRNA-specific DNA-oligo- nucleotide (RT6-’miRNA’ or RT6-’snRNA’ and the RevertAid™ M-MuLV Reverse Transcriptase (Fermentas GmbH) were employed to transcribe miRNAs and refer- ence snRNAs into cDNA. The RT-6-Pool represented a blend of DNA-oligonucleotides specific for 26 miRNAs as well as 5 snRNAs serving as reference genes for nor- malisation. The final concentration of the RT-6-Pool was