Limitations of mRNA amplification from small-size cell samples

BACKGROUND: Global mRNA amplification has become a widely used approach to obtain gene expression profiles from limited material. An important concern is the reliable reflection of the starting material in the results obtained. This is especially important with extremely low quantities of input RNA where stochastic effects due to template dilution may be present. This aspect remains under-documented in the literature, as quantitative measures of data reliability are most often lacking. To address this issue, we examined the sensitivity levels of each transcript in 3 different cell sample sizes. ANOVA analysis was used to estimate the overall effects of reduced input RNA in our experimental design. In order to estimate the validity of decreasing sample sizes, we examined the sensitivity levels of each transcript by applying a novel model-based method, TransCount. RESULTS: From expression data, TransCount provided estimates of absolute transcript concentrations in each examined sample. The results from TransCount were used to calculate the Pearson correlation coefficient between transcript concentrations for different sample sizes. The correlations were clearly transcript copy number dependent. A critical level was observed where stochastic fluctuations became significant. The analysis allowed us to pinpoint the gene specific number of transcript templates that defined the limit of reliability with respect to number of cells from that particular source. In the sample amplifying from 1000 cells, transcripts expressed with at least 121 transcripts/cell were statistically reliable and for 250 cells, the limit was 1806 transcripts/cell. Above these thresholds, correlation between our data sets was at acceptable values for reliable interpretation. CONCLUSION: These results imply that the reliability of any amplification experiment must be validated empirically to justify that any gene exists in sufficient quantity in the input material. This finding has important implications for any experiment where only extremely small samples such as single cell analyses or laser captured microdissected cells are available

Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance

BACKGROUND: A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20–200 micro-grams total RNA or 0.5–2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material. RESULTS: Using analysis of variance (ANOVA) and multiple hypothesis testing, we estimated the impact of amplification on the preservation of gene expression ratios. Both methods showed that the gene expression ratios were not completely preserved between amplified and non-amplified material. We also compared the expression ratios between the two cell lines for the amplified material with expression ratios between the two cell lines for the non-amplified material for each gene. With the aid of multiple t-testing with a false discovery rate of 5%, we found that 10% of the genes investigated showed significantly different expression ratios. CONCLUSION: Although the ratios were not fully preserved, amplification may prove to be extremely useful with respect to characterizing low expressing genes

Causal modeling and inference for electricity markets

How does dynamic price information flow among Northern European electricity spot prices and prices of major electricity generation fuel sources? We use time series models combined with new advances in causal inference to answer these questions. Applying our methods to weekly Nordic and German electricity prices, and oil, gas and coal prices, with German wind power and Nordic water reservoir levels as exogenous variables, we estimate a causal model for the price dynamics, both for contemporaneous and lagged relationships. In contemporaneous time, Nordic and German electricity prices are interlinked through gas prices. In the long run, electricity prices and British gas prices adjust themselves to establish the equlibrium price level, since oil, coal, continental gas and EUR/USD are found to be weakly exogenous

Benefit and risk assessment of breastmilk for infant health in Norway: Opinion of the Steering Committee of the Norwegian Scientific Committee for Food Safety

Source at https://vkm.no/english/riskassessments/allpublications/benefitandriskassessmentofbreastmilkforinfanthealthinnorway.4.27ef9ca915e07938c3b2a6df.html.Breastmilk covers all the nutritional needs of the infant the first months of life, with the exception of vitamin D. In addition, breastmilk has a number of protective properties. However, as humans are exposed to polluting chemicals through food, air, water and skin contact, breastmilk also contains contaminants. Taking the present-day levels of contaminants in Norwegian breastmilk and the long duration of breastfeeding (12 months) in Norway into account, the Norwegian Scientific Committee for Food Safety concludes that contaminants poses a low risk to Norwegian infants, and that the benefits of breastmilk to Norwegian infants clearly outweigh the risk presented by contaminants

Estimating and decomposing total uncertainty for survey-based abundance estimates of Norwegian spring-spawning herring

We demonstrate how the total uncertainty of the abundance estimate and abundanceat- age estimate of Norwegian spring-spawning herring are affected by uncertainty in the parameters of vessel avoidance, shadowing and depth dependent target strength, and uncertainty from trawl hauls and spatial-temporal coverage. The total uncertainty is decomposed into the sum of the contribution from each source separately. In addition, we highlight the potentially dramatic combined effect of correcting for vessel avoidance, shadowing and depth dependent target strength on the abundance estimate. The main framework is believed to be a promising tool for focusing the effort for reducing the uncertainty in the abundance estimates. The method is applied to data from surveys on the over-wintering stock in the Vestfjord system and Vesteraalen in northern Norway in November/December in the years 2001–2004