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A computer programme for the calculation of radioactivity data from liquid-scintillation counters fitted with external standards.

ABSTRACT: 1. A method for transferring data from liquid-scintillation counters to automatic punched-card read-out is described. 2. A method of calibration of an external standard is presented that supplies sufficient information for the correction of quenched data by computer. 3. A computer programme in FORTRAN IV is described that calculates the data from one isotope, or two isotopes counted simultaneously, and that incorporates features for examining the reproducibility of the input data. 4. With the methods described, quench correction of samples containing either (3)H or (14)C was accurate until samples were quenched 70%. When the two isotopes were both present in one sample and counted simultaneously, the correction was accurate until the samples were quenched 60%. When quenching was greater than either 70 or 60% respectively, its correction was still possible but with slightly diminished accuracy and with greater variability.

SUBMITTER: Felts JM

PROVIDER: S-EPMC1198372 | biostudies-other | 1967 Nov

REPOSITORIES: biostudies-other

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Project description:Acute cellular stress is known to induce a global reduction in protein translation through suppression of cap dependent translation. However, selective translation in response to acute stress has been shown to play important roles in regulating the stress response. An accurate transcriptome-wide profile of acute cellular stress-induced translational changes has been challenging to obtain. Commonly used data normalization methods, such as quantile normalization, operate based on the assumption that any systematic shifts are artifacts introduced from experimental procedures. Consequently, if applied to profiling acute cellular stress-induced protein translation changes, these methods are expected to produce biased estimates. To address this issue, here we designed, generated, and evaluated a panel of 16 oligomers to serve as external standards for ribosome profiling studies. Using Sodium Arsenite treatment-induced oxidative stress in lymphoblastoid cell lines as a model system, we applied spike-in oligomers as external standards based on quantifications of monosomal RNA extracted from each sample. We found our spike-in oligomers to display a linear correlation between the observed and the expected, with small but significant ratio compression at the lower concentration range, and span the expected quantitative range in the observed data, which covers 97 % of the quantitated endogenous genes. We found popular global scaling normalization approaches to introduce both high levels of false positives and false negatives in differential expression analysis. Using the expected fold changes constructed from spike-in external controls, we found in our dataset that TMM normalization produced 87.5% false positives when a P value cutoff of 0.1 is used (i.e. 10% expected false positive rate) and on average produced a systematic shift of fold change by 3.25 fold. These results highlight the consequences of applying global scaling approaches to conditions that clearly violate their key assumptions. As an alternative, we found using spike-in quantifications as control genes in RUVg normalization recapitulated the expected stress induced global reduction of translation, and resulted in little, if any, systematic shifts in spike-in constructed true positives. Finally, using spike-in constructed true positives and true negatives, we explored alternative normalization approaches for acute cellular stress response ribo-seq studies. We found that a simple approach that quantile normalized data from control and treated samples separately, which we termed respective quantile normalization, produced expected results in spike-in quantification, and resulted in little, if any, systematic bias on fold change in endogenous genes. Additionally, we found that under certain parameters, using endogenous control genes for RUVg normalization best recapitulate the expected. Our results clearly demonstrated the utility of our spike-in oligomers, both for constructing expected results as controls and for data normalization. Our exploration of different normalization approaches highlights the issues in applying global scaling normalization when key assumptions are clearly not met. We show that a respective quantile normalization approach or normalization with endogenous control genes are viable options worth considering as a more generalizable approach for stress response ribo-seq studies. This conclusion is likely applicable to other types of studies that involve global shifts in expression profiles between comparison groups of interests.

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A computer programme for the calculation of radioactivity data from liquid-scintillation counters fitted with external standards.

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