Ves were generated by a cycle of 95uC for 1 min, 65uC for 1 min and 80 cycles starting at 65uC with 0.5uC increments.RNA extraction, analysis of RNA preparation, and cDNA synthesisOnce 10781694 thawed, NP Th those from patients, but again there was no correlation with samples (200 ml) had 1 volume of RNAprotect BacteriaH (Qiagen) added and were immediately centrifuged for 15 min at 150006g in a refrigerated centrifuge (Eppendorf). Total RNA was then extracted from the pellet using the RNeasy Mini Kit (Qiagen) as outlined by the manufacturer and additionally treated with 2 U of DNaseI (Promega) essentially as previously described [31,40]. Integrity of our RNA preparations [RNA integrity number (RIN)], and RNA concentration of samples, were obtained by using the RNA 6000 Nano kit or RNA 6000 Pico kit electrophoresis system and the 2100 Bioanalyzer (Agilent technologies). Total RNA (500 pg) was cDNA transcribed using the iScript cDNA synthesis kit (Bio-Rad) and following the manufacturer’s instructions.Development and validation of a panel of primers to amplify S. pneumoniae genesThe challenge to detect S. pneumoniae genes by PCR, or their transcripts by RT-PCR, in NP samples relies on the potential presence of homologous sequences in species sharing the human upper respiratory tract (including the nasopharynx) with the pneumococcus [41]. Therefore, we first designed and validated, a panel of 21 16985061 pair of primers (Table 1) to amplify genes encoding known virulence and colonization factors in the pneumococcus (i.e. ply, nanA, iga, etc.), adhesins (i.e. pavA, pspA, rrgB, etc.) or genes linked to regulatory functions (i.e. luxS, comC, mgrA and codY) [1,9]. These primers were designed based on sequences available from reference S. pneumoniae strains D39 and TIGR4 [37,42]. Bioinformatic analysis of both forward and reverse primer sequences revealed that, with some exceptions listed below, most of these Table 1 pair of primers would only hybridize S. pneumoniae sequences. Besides analyzing each primers individually, both forward (i.e. 22 bp) and reverse sequences (i.e. 22 bp) were also placed together (i.e. 44 bp) and then analyzed again using BLAST. The criteria to define in silico hybridization of these concatenated sequences included query coverage .70 and E-score ,0.1. Genes/genomes meeting these criteria were individually analyzed to confirm that both primers (i.e. forward and reverse) hybridizedQuantitative RT-PCR (qRT-PCR)Reactions were performed as described above except that in qRT-PCR reactions we utilized 2 ml of cDNA as template. For the relative quantification of mRNA molecules, purified genomic DNA from S. pneumoniae reference strain TIGR4 or D39 was serially diluted to prepare standards representing 2.146101, 4.296101, 4.296102, 4.296103, 4.296104, or 4.296105 genomeExpression of Sp Genes in the Human Synthesis is positively regulated by iron stores and Tf saturation, the Nasopharynxwithin the same gene. For example, Fig. 2 shows that the gene encoding enolase (eno) had a perfect match with S. pneumoniae strain ST556 (query coverage of 100 and an E-score of 0.004). Both forward and reverse sequences were identified within the same (eno) gene (Fig. 2, left bottom panel). Conversely, query coverage of 78 with an E-score of 0.004, which would comply with our criteria for in silico hybridization, was detected in S. pyogenes strain MGAS1882. However, only 24 bp hybridized in a putative eno gene whereas another 17 bp fragment hybridized elsewhere in the genome (Fig. 2, right bottom panel). This in silico hybridization was, therefore, not compatible with a potential PCR product.Ves were generated by a cycle of 95uC for 1 min, 65uC for 1 min and 80 cycles starting at 65uC with 0.5uC increments.RNA extraction, analysis of RNA preparation, and cDNA synthesisOnce 10781694 thawed, NP samples (200 ml) had 1 volume of RNAprotect BacteriaH (Qiagen) added and were immediately centrifuged for 15 min at 150006g in a refrigerated centrifuge (Eppendorf). Total RNA was then extracted from the pellet using the RNeasy Mini Kit (Qiagen) as outlined by the manufacturer and additionally treated with 2 U of DNaseI (Promega) essentially as previously described [31,40]. Integrity of our RNA preparations [RNA integrity number (RIN)], and RNA concentration of samples, were obtained by using the RNA 6000 Nano kit or RNA 6000 Pico kit electrophoresis system and the 2100 Bioanalyzer (Agilent technologies). Total RNA (500 pg) was cDNA transcribed using the iScript cDNA synthesis kit (Bio-Rad) and following the manufacturer’s instructions.Development and validation of a panel of primers to amplify S. pneumoniae genesThe challenge to detect S. pneumoniae genes by PCR, or their transcripts by RT-PCR, in NP samples relies on the potential presence of homologous sequences in species sharing the human upper respiratory tract (including the nasopharynx) with the pneumococcus [41]. Therefore, we first designed and validated, a panel of 21 16985061 pair of primers (Table 1) to amplify genes encoding known virulence and colonization factors in the pneumococcus (i.e. ply, nanA, iga, etc.), adhesins (i.e. pavA, pspA, rrgB, etc.) or genes linked to regulatory functions (i.e. luxS, comC, mgrA and codY) [1,9]. These primers were designed based on sequences available from reference S. pneumoniae strains D39 and TIGR4 [37,42]. Bioinformatic analysis of both forward and reverse primer sequences revealed that, with some exceptions listed below, most of these Table 1 pair of primers would only hybridize S. pneumoniae sequences. Besides analyzing each primers individually, both forward (i.e. 22 bp) and reverse sequences (i.e. 22 bp) were also placed together (i.e. 44 bp) and then analyzed again using BLAST. The criteria to define in silico hybridization of these concatenated sequences included query coverage .70 and E-score ,0.1. Genes/genomes meeting these criteria were individually analyzed to confirm that both primers (i.e. forward and reverse) hybridizedQuantitative RT-PCR (qRT-PCR)Reactions were performed as described above except that in qRT-PCR reactions we utilized 2 ml of cDNA as template. For the relative quantification of mRNA molecules, purified genomic DNA from S. pneumoniae reference strain TIGR4 or D39 was serially diluted to prepare standards representing 2.146101, 4.296101, 4.296102, 4.296103, 4.296104, or 4.296105 genomeExpression of Sp Genes in the Human Nasopharynxwithin the same gene. For example, Fig. 2 shows that the gene encoding enolase (eno) had a perfect match with S. pneumoniae strain ST556 (query coverage of 100 and an E-score of 0.004). Both forward and reverse sequences were identified within the same (eno) gene (Fig. 2, left bottom panel). Conversely, query coverage of 78 with an E-score of 0.004, which would comply with our criteria for in silico hybridization, was detected in S. pyogenes strain MGAS1882. However, only 24 bp hybridized in a putative eno gene whereas another 17 bp fragment hybridized elsewhere in the genome (Fig. 2, right bottom panel). This in silico hybridization was, therefore, not compatible with a potential PCR product.