The stability of standard gene expression is an elementary prerequisite for internal standardisation of target gene expression data and many so called housekeeping genes with assumed stable expression can exhibit either up- or down-regulation under some experimental conditions. The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index. The index can be compared with further ten target genes to decide, whether they are differentially expressed under an applied treatment. All data processing is based on crossing points. The BestKeeper software tool was validated on four housekeeping genes and 10 members of the somatotropic axis differentially expressed in bovine corpora lutea total RNA. The BestKeeper application and necessary information about data processing and handling can be downloaded on http://www.wzw.tum.de/gene-quantification/bestkeeper.html.

Summary Background Ending the global tobacco epidemic is a defining challenge in global health. Timely and comprehensive estimates of the prevalence of smoking tobacco use and attributable disease burden are needed to guide tobacco control efforts nationally and globally. Methods We estimated the prevalence of smoking tobacco use and attributable disease burden for 204 countries and territories, by age and sex, from 1990 to 2019 as part of the Global Burden of Diseases, Injuries, and Risk Factors Study. We modelled multiple smoking-related indicators from 3625 nationally representative surveys. We completed systematic reviews and did Bayesian meta-regressions for 36 causally linked health outcomes to estimate non-linear dose-response risk curves for current and former smokers. We used a direct estimation approach to estimate attributable burden, providing more comprehensive estimates of the health effects of smoking than previously available. Findings Globally in 2019, 1·14 billion (95% uncertainty interval 1·13–1·16) individuals were current smokers, who consumed 7·41 trillion (7·11–7·74) cigarette-equivalents of tobacco in 2019. Although prevalence of smoking had decreased significantly since 1990 among both males (27·5% [26·5–28·5] reduction) and females (37·7% [35·4–39·9] reduction) aged 15 years and older, population growth has led to a significant increase in the total number of smokers from 0·99 billion (0·98–1·00) in 1990. Globally in 2019, smoking tobacco use accounted for 7·69 million (7·16–8·20) deaths and 200 million (185–214) disability-adjusted life-years, and was the leading risk factor for death among males (20·2% [19·3–21·1] of male deaths). 6·68 million [86·9%] of 7·69 million deaths attributable to smoking tobacco use were among current smokers. Interpretation In the absence of intervention, the annual toll of 7·69 million deaths and 200 million disability-adjusted life-years attributable to smoking will increase over the coming decades. Substantial progress in reducing the prevalence of smoking tobacco use has been observed in countries from all regions and at all stages of development, but a large implementation gap remains for tobacco control. Countries have a clear and urgent opportunity to pass strong, evidence-based policies to accelerate reductions in the prevalence of smoking and reap massive health benefits for their citizens. Funding Bloomberg Philanthropies and the Bill & Melinda Gates Foundation.

We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1–16 qPCR replicates per concentration and we tested 2–10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.

We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.

Genetic variability in the promoter and 3' region of the SNCA gene coding alpha-synuclein modulates the risk to develop sporadic Parkinson's disease (PD). Whether this is mediated by regulating alpha-synuclein expression levels remains unknown. Therefore, we analyzed levels of alpha-synuclein in blood and human post mortem brain tissue including the substantia nigra using quantitative real-time reverse transcriptase-polymerase chain reaction and enzyme linked immunosorbent assay in vivo. Single nucleotide polymorphism (SNP) rs356219, a tagging SNP for a disease-associated haplotype in the 3' region of the SNCA gene, has a significant effect on SNCA mRNA levels in the substantia nigra and the cerebellum. Further, the "protective" genotype 259/259 of the PD-associated promoter repeat NACP-Rep1 is associated with lower protein levels in blood than genotypes 261/261, 259/261, and 259/263. In conclusion, we provide evidence that alpha-synuclein levels are influenced by genetic variability in the promoter and 3' region of the SNCA gene in vivo.