DOI: 10.15482/usda.adc/28836296.v1
title: Data from: Quantification and diagnostic relevance of blood and heme-mediated inhibition of prion detection by RT-QuIC

description: Purpose of the study:The mechanisms of inhibition mediated by heme on the prion protein misfolding reaction and detection of scrapie prions by the real-time quaking-induced conversion (RT-QuIC) assay. Heme was studied as free hemin, as heme within hemoglobin (Hb), and as present in lysed whole blood. Experiments were conducted to determine the sensitivity of the assay to each of these sources of heme (Fig1), the time-dependent direct effect of blood or Hb on the prion seed prior to analysis (Fig4), the sensitivity of the assay to the concentration of recombinant prion protein substrate (rPrP) in the reaction mixture (Fig7), and the inhibitory effects Hb in holo- and apo- forms (Fig9).The repository files:RT-QuIC reaction dataEach RTQuIC_reactions_Fig_#.csv file provides the RT-QuIC reaction data for each experiment as identified by its corresponding Figure number (Fig 1, 4, 7, or 9) from the original manuscript. Plots of these raw data are also provided as Fig #.png files.The first row in each worksheet names the variables (columns). Depending on the specific experiment, the variables may include the following.Merge_file: The auto numbered file name of the data from one 96-well plate.well: A single well identified by its row (letter) and column (number) position in a 96-well plate.hours: The elapsed decimal hours of an experiment.seed_dilution_factor: The factor of a 10-fold dilution series. For example, a seed dilution of 3 is the third 10-fold dilution of the prion seed. Thus, dilution of the original sample 1,000 times.h_type: The form of heme applied--as hemin, hemoglobin, or as blood (lysed whole blood). PBS represents the control condition with zero added heme in any form.h_conc: The final concentration of heme (µg/ml).experiment: (Only relevant to Fig 4.) Identifies the exposure experiment per 96-well plate as blood or hemoglobin (Hb) exposure of the seed prior to analysis.incubation_d: (Only relevant to Fig 4.) The number of days exposure to blood, hemoglobin (Hb), or PBS.substrate_conc: (Only relevant to Fig 7.) The final concentration of rPrP (mg/ml) in the reaction mixture.Hb_type: (Only relevant to Fig 9.) The form of hemoglobin (Hb) applied--in holo form (Hb) or as stripped of heme (apoHb). PBS represents the control condition with zero added Hb in either form.Hb_conc: (Only relevant to Fig 9.) The final concentration of hemoglobin (Hb) as measured by heme content (µg/ml).Fig 1.pngGraphs of RT-QuIC reactions demonstrating the concentration-dependent effects of heme on detection of scrapie prion seeds in samples of sheep hindbrain. Heme was added to the reaction mixture either as free hemin, as heme within hemoglobin, or as found in lysed whole blood. The relative effects of these heme forms were tested over a 3 to 6 10-fold dilution range of the hindbrain sample (thus, of the prion seeds). Each graph window includes the color-coded replicate well reactions. y-axis: thioflavin T (ThT) measured in relative fluorescence units. x-axis: assay time in decimal hours (0 to 100 hours).Fig 4.pngGraphs of RT-QuIC reactions demonstrating the time-dependent effects of prion seed exposure to either hemoglobin (Hb) or lysed whole blood prior to analysis. In each experiment, PBS was substituted as the comparative control. After 1- or 7-days seed exposure, the sensitivity of the assay for each pre-exposure condition was tested by limiting 10-fold dilution. Each graph window includes the color-coded replicate well reactions. y-axis: thioflavin T (ThT) measured in relative fluorescence units. x-axis: assay time in decimal hours (0 to 100 hours).Fig 7.pngGraphs of RT-QuIC reactions demonstrating the dependence of reactions on the concentration of substrate recombinant prion protein (rPrP) in the reaction mixture. Each graph window includes the color-coded replicate well reactions. y-axis: thioflavin T (ThT) measured in relative fluorescence units. x-axis: assay time in decimal hours (0 to 100 hours).Fig 9.pngGraphs of RT-QuIC reactions comparing the concentration-dependent effects of hemoglobin in the reaction mixture either as native hemoglobin (Hb, holo- form) or in its apo- form (apoHb) in which the heme moiety has been stripped away. \In each experiment, PBS was substituted as the comparative control. The relative effects of Hb in these forms were tested at 4 and 5 10-fold dilutions of the hindbrain sample (i.e., of the prion seeds). Each graph window includes the color-coded replicate well reactions. y-axis: thioflavin T (ThT) measured in relative fluorescence units. x-axis: assay time in decimal hours (0 to 100 hours).Supplementary figure filesFig S1.pdf Factors that affect baseline ThT Fluorescence in RT-QuIC. <i>Caption and authors' comment text included in file.</i>Fig S2.pdfDifferential spectra and spectral shifts resulting from inhibitor interactions with rPrP in RT-QuIC buffer. <i>Caption text included in file.</i>Fig S3.pdfUnseeded RT-QuIC assay with 0.10, 0.15, and 0.20 mg/ml rPrP in the presence of inhibitory Hb concentrations.