For today's academic research labs, the new Roche LightCycler 96 System is the optimal mid-size, mid-throughput real-time PCR solution that conveniently delivers the highest standards expected from a LightCycler System: An ideal combination of accuracy, temperature homogeneity, and reproducibility, matched with powerful software so intuitive that it is accessible to any user.
Up to 4 channels, pre-calibrated color compensation (no user interaction necessary) All channels can be viewed by the software in one chart simultaneously or viewed in 4 charts separately on one screen
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We next examined whether complex I subunits were also depleted in human oocytes. Early oocytes and ovarian somatic cells were isolated from ovarian cortices of patients, and analysed by label-free proteomics. We identified 40% of all known mitochondrial proteins (Supplementary Table 3). The upregulation of proteins related to UPRmt was conserved in human early oocytes, and further confirmed with immunofluorescence (Fig. 3c and Extended Data Fig. 4b). An analysis of the OXPHOS machinery comparing oocytes and ovarian somatic cells revealed that, in line with the Xenopus data, many complex I subunits were either at very low levels or not identified in human oocytes (Fig. 3d,e and Extended Data Fig. 5a).
Label-free samples were acquired in data-dependent acquisition mode and full MS scans were acquired in the Orbitrap. In each cycle of data-dependent acquisition analysis, the most intense ions were selected for fragmentation. Fragment ion spectra were produced through high-energy collision dissociation at a normalized collision energy of 28%, and they were acquired in the ion trap mass analyser.
A third method, High Resolution Melting (HRM) analysis, is also available on the LightCycler 480 System after installation of a separate software module, the LightCycler 480 Gene Scanning Software. Although its use for genotyping has been widely documented in the literature, it is recommended for mutation discovery rather than detection/genotyping purposes.
Figure 4: 96 different samples of total human genomic DNA (10 ng/µl) were applied to four adjacent wells of a 384 multiwell plate (e.g., sample #1 to wells A1, A2, B1, B2). Primers and SimpleProbe probes allowing the detection of four different SNPs were added to the wells in a checkerboard pattern, allowing the genotyping of all SNPs in all samples simultaneously. For all studied SNPs, both homozygous as well as heterozygous samples were found. Results are shown based on the use of the checkerboard subset feature included in the analysis software. MDR: multidrug resistance protein; LPLH: lipoprotein lipase H; ADD:adenosine deaminase; ADR: adrenergic receptor
According to the current literature, exploring putative reference genes from the transcriptomic data is a feasible and easy assay in fungal species (Llanos et al. 2015; Nadai et al. 2015; Steiger et al. 2010). Likewise, there is a series of popular Excel-based software programs, such as geNorm, NormFinder and BestKeeper, that have been successfully used in previous studies (Vandesompele et al. 2009; Andersen et al. 2004; Pfaffl et al. 2004).
To select suitable reference genes, three software packages were used to calculate the stability: NormFinder, geNorm, and BestKeeper. An additional web-based tool, RefFinder, ( ) was applied to integrate and rank all candidate reference genes (Xie et al. 2012).
Three different applets, geNorm, NormFinder, and BestKeeper, were applied to measure and rank the stability of candidate reference genes. The program geNorm (Steiger et al. 2010) classifies genes according to the control gene stability measure (M value), which represents the average of the pair-wise variation of a gene with all other control genes. NormFinder (Vandesompele et al. 2009) examines the stability of each single candidate gene independently and not in relation to the other genes. The results from geNorm and NormFinder can be compared easily because they both use raw data (relative quantities) as input data. BestKeeper (Andersen et al. 2004) is another Excel-based tool that determines the optimal reference genes using a pair-wise correlation analysis (Pearson correlation coefficient) of all pairs of candidate genes. It uses CP values (instead of relative quantities) as input and employs a different measure of expression stability from geNorm and NormFinder. However, this software is not able to analyse more than ten reference genes together, and thus the first ten ranking genes should depend on geNorm and NormFinder. The rankings of these software programs relied on different algorithms and were expected to lead to distinct outputs. Therefore, another software program, RefFinder (Radonić et al. 2004), was used to integrate the currently available major computational programs (geNorm, NormFinder, BestKeeper, and the comparative ΔΔCt method) to compare and rank the tested candidate reference genes. Based on the rankings from each program, it could assign an appropriate weight to an individual gene and calculate the geometric mean of their weights for the overall final ranking.
Mycobacterium avium subsp. paratuberculosis (MAP) is a well-known causative agent of paratuberculosis, a chronic infectious granulomatous enteritis of ruminants contributing to significant economic losses worldwide. Current conventional diagnostic tools are far from being sufficient to manage and control this disease. Therefore, increased attention has been paid to alternative approaches including phage-based assays employing lytic bacteriophage D29 to detect MAP cells. The aim of the present study was to assess the applicability and efficiency of the recently developed phage-based kit termed Actiphage combined with IS900 real-time PCR (qPCR) for rapid detection and quantification of viable MAP in milk samples. We demonstrated that Actiphage in combination with IS900 qPCR allows for rapid and sensitive detection and identification of viable MAP in milk samples with a limit of detection of 1 MAP per 50 ml milk. Using this method, the presence of viable MAP cells was successfully determined in 30.77% of fresh goat, sheep and cow milk samples originating from paratuberculosis-affected herds. We further used Actiphage assay to define the time-lapse aspect of testing naturally contaminated milk and milk filters frozen for various lengths of time by phage-based techniques. Viable MAP was detected in 13.04% of frozen milk samples and 28.57% of frozen milk filters using Actiphage-qPCR. The results suggest the ability to detect viable MAP in these samples following freezing for more than 1 year. The obtained results support the views of the beneficial role of this technology in the control or monitoring of paratuberculosis.
Proteins were separated by 10-12% SDS-PAGE and transferred onto PVDF membrane (Immobilon-P membrane; Millipore; Bedford, MA, USA). Membranes were incubated with specific antibodies, followed by washing and incubation with a horseradish peroxidase-conjugated secondary antibody. Specific antibody-antigen complex was detected using an enhanced chemiluminescence Western Blot detection system (Thermo Fisher Scientific Inc.). The amount of detected proteins was quantified by ImageJ software (NIH, Bethesda, MD, USA), and was expressed as the ratio to β-actin protein. Each assay was performed in quadruplicates.
While most quantitative PCR machines have the option of melting curve generation and analysis, the level of analysis and software support varies. High Resolution Melt (known as either Hi-Res Melting, or HRM) is the advancement of this general technology and has begun to offer higher sensitivity for SNP detection within an entire dye-stained amplicon. It is less expensive and simpler in design to develop probeless melting curve systems. However, for genotyping applications, where large volumes of samples must be processed, the cost of development may be less important than the total throughput and ease of interpretation, thus favoring probe-based genotyping methods.
Human chronic myeloid leukaemia and breast cancer cell lines, used in this study, were obtained from the American Type Culture Collection (ATCC) and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Cells were grown in T225 tissue culture flasks in RPMI1640 media (phenol red free; Sigma) supplemented with 10% foetal calf serum and 1% L-Glutamine (Life Technologies Bethesda Research Laboratories Ltd) and maintained at 37 C with 5% CO2. Each cell line was passaged up to 3 times and at approximately 80% confluence, the cells were fixed in 10% neutral buffered formalin for 24 h at room temperature before embedding in paraffin wax. Consecutive 5 μm sections were generated from each of the FFPE cell blocks using a standard microtome blade and fixed onto glass microscope slides. All FFPE cell blocks and 5 μm sections were stored at room temperature until nucleic acid extraction was performed.
Graphical representations were generated using TIBCO Spotfire software version 5.0 and Microsoft Office Excel 2007. Data was represented as the geometric mean with corresponding geometric standard error as the data was found to have an asymmetrical distribution. The statistical significance of the data was assessed using two sample unequal variance t tests as preliminary analysis was performed in the absence of an appropriate rationale as to the direction of the relationship. In the results and discussion, we refer to P-values 2ff7e9595c
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