An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.

Quantitative detection of human enteric adenoviruses in river water by microfluidic digital polymerase chain reaction.

We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.

Decomposition of waste DNA with extended autoclaving under unsaturated steam.

Carryover and false-positive amplification of undesired DNA sequences are serious problems in research and diagnostic testing using PCR. One possible source of DNA cross-contamination can be the autoclave if DNA contained in waste is not effectively decomposed and contaminates the autoclave. To assess this possibility, we used a 2682 bp PCR product as a model waste DNA and quantified the amplifiability of an 84 bp short fragment derived from the model waste DNA in the steam and the residual bottom water after autoclaving. Autoclaving under the standard conditions of 121°C for 20 min did not sufficiently remove amplifiability from the model DNA and was found to be a possible source of laboratory contamination. However, the amplifiable template was removed after autoclaving at 121°C for 80 min. Fragmentation and hydrolysis may occur during autoclaving, and the presence of atmospheric oxygen facilitated the decomposition. These findings will help researchers develop better strategies for disposing of DNA waste.

Toward metrological traceability for DNA fragment ratios in GM quantification. 1. Effect of DNA extraction methods on the quantitative determination of Bt176 corn by real-time PCR.

An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.

Changes in phosphorus removing performance and bacterial community structure in an enhanced biological phosphorus removal reactor.

A lab-scale-enhanced biological phosphorus removal (EBPR) reactor was operated for 204 days to investigate the correlation between phosphorus removing performance and bacterial community structure. The phosphorus removing performance was good from day 1 to 92 and from day 172 to 204. However, the removal activity was in a deteriorated state from day 93 to 171. From day 69 (2 weeks before the beginning of the deterioration) to 118 (2 weeks after the beginning of the deterioration), sludge P content decreased. The amounts of ubiquinone-8 and menaquinone-8 (H(4)) decreased during this period while the amount of ubiquinone-10 increased. The comparison of these changes and the general attribution of each quinone to the bacterial phylogenetic groups suggested that beta proteobacteria and Actinobacteria contributed to EBPR positively, and that alpha proteobacteria were related to this EBPR deterioration. Glycogen accumulating organisms (GAOs) are considered to detrimentally affect EBPR ability by outcompeting the phosphorus accumulating organisms by using aerobically synthesized glycogen as the energy source to assimilate organic substrates anaerobically to form polyhydroxyalkanoates. However, in this research, there was nearly no substrate uptake during the anaerobic period at the middle of the deteriorated performance period. This suggests that the deterioration observed in this research does not agree with the GAOs inhibition model. In this research, the excess P release at the anaerobic period was concluded to cause the deterioration.