Evaluation of the M RT-PCR assay with clinical samples.

<p>A representative set of clinical samples was simultaneously tested for the BCSP31 gene for <i>Brucella</i> spp (A) and the intergenic region SenX3-RegX3 for <i>M. tuberculosis</i> complex (B). Panel (A) Samples 1, 2 and 3 were pleural fluid, hepatic abscess and urine, respectively, corresponding to brucellosis patients; sample 7 was CSF, from a patient with <i>S. pneumoniae</i> meningitis; and samples 4 and 5, positive controls for <i>Brucella</i> and <i>M tuberculosis</i> complex, respectively. Sample 6, negative control. Panel (B). Samples 1, 2 and 3 were lymph node, pericardial tissue and psoas abscess, respectively, corresponding to tuberculosis patients; sample 7 was of vertebral tissue, from a patient with <i>S. agalatiae</i> pyogenic vertebral osteomyelitis; and samples 4 and 5, positive controls for <i>M. tuberculosis</i> complex and <i>Brucella</i>, respectively. Sample 6, negative control. Panels (C) and (D). Melting curves of the amplified fragments generated by M RT-PCR. Specific signals for brucellosis patients and positive controls had melting temperatures of 71.51±0.18°C and 71.12±0.13°C for tuberculosis patients and positive controls.</p

Multiplex PCR results with DNA from different <i>Brucella</i> and <i>Mycobacterium</i> strains.

<p>FMV, Facultad de Medicina Valladolid, Valladolid, Spain; CAJA, Consejeria de Agricultura, Junta de Andalucia, Seville, Spain; CECT, Colección Española de Cultivos Tipo, Valencia, Spain; HCH, Hospital Carlos Haya, Málaga, Spain. ATCC, American Type Culture Collection.</p

Nucleotide sequences and positions of primers and probes for amplification and detection of <i>Brucella</i> spp. and <i>M. tuberculosis</i> complex for M RT-PCR.

<p>Red 640- LightCycler Red 640, Red 705- LightCycler Red 705, FL-5,6-carboxifluoresceína.</p

Diagnostic yield of Multiplex real-Time PCR in clinical specimens from patients with extrapulmonary tuberculosis and focal complications of brucellosis.

<p>PPV = positive predictive value; NPV = negative predictive value; Positive LR = positive likelihood ratio, Negative LR = negative likelihood ratio.</p>a<p>not done for mathematical reasons (division by zero).</p

Double-tube format RT-PCR assay with identical clinical samples to those described in Figure 2.

<p>All reactions were optimized to obtain the best amplification kinetics under the same cycling conditions and reaction mixture compositions, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004526#s2" target="_blank">Methods</a> section. Panel (A) Clinical samples tested for the BCSP31 gene for <i>Brucella</i> spp. Samples 1, 2 and 3 were pleural fluid, hepatic abscess and urine, respectively, corresponding to brucellosis patients. Sample 4 was CSF, from a patient with <i>S. pneumoniae</i> meningitis; and samples 5, 6 and 7 were lymph node, pericardial tissue and psoas abscess, respectively, corresponding to tuberculosis patients, and sample 8 was of vertebral tissue from a patient with <i>S. agalatiae</i> pyogenic vertebral osteomyelitis. Samples 9 and 10, positive controls for <i>Brucella</i>. Sample 11, negative control. Panel (B). Clinical samples assayed for the intergenic region SenX3-RegX3 for <i>M. tuberculosis</i> complex, Samples 1 to 8 were identical to those described in panel A. Samples 9 and 10, positive controls for <i>M. tuberculosis</i> complex. Sample 11, negative control. Panels (C) and (D). Melting curves of the amplified fragments generated by RT-PCR in double-tube format.</p

The table shows the primer pairs used for each gene for mice.

<p>The primer pairs included a control region of the PPARG gene to evaluate the correct performance of the IP. Abbreviations: Transcription start site (TSS); Nucleotides (nt).</p

The chart shows the different efficiency in total DNA recovery between the Dounce and the Ultraturrax homogenizer.

<p>100 mg of frozen adipose tissue was fixed in 1% paraformaldehyde, homogenized either using the Dounce or Ultraturrax homogenizer and sheared for 40 cycles (30 seconds ON and 30 seconds OFF). A sample of 50 μl of the homogenized material was then taken, and the chromatin was de-crosslinked using the fast Chelex-100 method. Total DNA was extracted and quantified by nanodrop. Data are given as means with error bars. Abbreviations: Dounce, Dounce homogenization method; Ultraturrax, ultraturrax homogenization method. (n = 6).</p

The table shows the primer pairs used for each gene for humans.

<p>The primer pairs included a control region of the SCD gene to evaluate the correct performance of the IP. Abbreviations: Transcription start site (TSS); Nucleotides (nt).</p

Chromatin immunoprecipitation improvements for the processing of small frozen pieces of adipose tissue - Fig 1

<p>The chart shows the three different workflows performed, using the pestle and mortar (1.1), the Dounce homogenizer (1.2) or the Ultraturrax homogenizer (1.3). In (1.1), the homogenization step was performed using liquid nitrogen, after which it was fixed. After this, the nuclei were pelleted and nucleus lysis buffer was added. Once incubated, the sample was sheared and the chromatin fragmentation and recovery were checked. In the other two alternative methods proposed (1.2 and 1.3), the tissue was cut in small pieces (3 mm) and the fixation step was performed prior to the homogenization. Once homogenized, the nuclei were recovered by centrifugation, nucleus lysis buffer was added and the sample was sheared and the chromatin fragmentation and recovery were checked.</p

100 mg of frozen adipose tissue was fixed in either 1% or 0.5% of paraformaldehyde and then homogenized using the Ultraturrax method.

<p>It was then sheared for 40 cycles (30 seconds ON and 30 seconds OFF), after which a sample of 50 μl of the homogenized material was taken, and the chromatin was de-crosslinked using the fast Chelex-100 method. Total DNA was extracted and quantified by nanodrop. Fixation at 0.5% presents higher levels of DNA recovery (A) after DNA purification and a better chromatin shear tested by electrophoresis in 2% agarose gel. (B) Comparison of the use of PBS+1% or PBS+0.5% formaldehyde in the performance of DNA recovery after de-crosslinking and purifying the DNA. (n = 6). Data are given as means with error bars.</p