Next, in order to identify differentially expressed genes, the SAM (Significance Analyses of Microarray) statistical package was CHIR-99021 in vitro used to compare the levels of gene expression among the following groups: (1) uninfected C57BL/6 and CBA macrophages; (2) L. amazonensis-infected C57BL/6 macrophages and uninfected cells; (3) L. amazonensis-infected CBA macrophages and uninfected cells; (4)
L. amazonensis-infectedC57BL/6 and CBA macrophages. In order to enhance confidence in the statistical analysis of microarray data, experiment variables of incubation and infection time were not considered when comparing gene expression among groups (1) to (4). SAM software uses a modified t-test measurement which corrects for
multiple comparisons by means of a False Discovery Rate (FDR) approach . The q-values, or the minimum FDRs at which a statistical test may be called significant , have been provided for each selleck chemicals differentially expressed gene in Tables S1, S2 and S3 (See Additional file 1: Table S1; Additional file 2: Table S2 and Additional file 3: Table S3, respectively). Finally, differentially expressed genes were analyzed and grouped in functional networks using the Ingenuity Pathway Analysis program v8.8 (IPA-Ingenuity Systems®, http://www.ingenuity.com). Possible networks and pathways were scored and modeled considering the sets of differentially expressed genes triclocarban derived from the four comparisons described above. To calculate the probability of associations between genes from the functional networks and pathways generated by IPA®, Fisher’s exact test was used with a 0.05 threshold value. Total macrophage mRNA extraction and mRNA quantification by RT-qPCR In order to perform reverse transcriptase-quantitative polymerase chain reactions (RT-qPCR), RNA was initially extracted from uninfected or infected macrophages using a QIAGEN Mini Kit (RNAeasy) in accordance
with manufacturer Entospletinib order directions. An optical density reading was taken following extraction procedures and RNA integrity was verified using an agarose gel. Complementary DNA (cDNA) was synthesized by reverse transcription in a final volume of 20 μL containing 5 mM MgCl2 (Invitrogen), PCR buffer 1× (Invitrogen), deoxyribonucleotide triphosphates each at 1 mM (dNTPs – Invitrogen), 0.5 mM oligonucleotide (oligo d(T) – Invitrogen), 1 UI RNase inhibitor (RNase Out – Invitrogen), 2.5 UI reverse transcriptase (MuLVRT- Invitrogen) and 1 μg of sample RNA in RNAse-Free Distilled Water. All reaction conditions consisted of a single cycle at 42°C for 50 min, followed by 70°C for 15 min and, finally, 4°C for at least 5 min. Following reverse transcription, the synthesized cDNA was aliquoted and frozen at -20°C. The cDNA aliquots were later thawed and amplified by qPCR in order to perform gene quantification.