Theme 4 Lecture 14 Flashcards
Why is it important to identify high-quality hits from screening?
Reduces time/waste and only selects active compounds
reliable and range
What is the significance of using positive and negative controls in screening assays?
Controls are critical for assessing reliability and determines the range of expected responses.
standard
What key parameters are used to evaluate the quality of a screening assay?
Signal, positive and negative control signals, background, mean, and standard deviation.
parameters
What are Signal-to-Noise and Z’ Factor, and why are they important?
Statistical parameters which asses assay quality
comparison
How are Signal-to-Noise and Z’ Factor used in practice?
They are implied to compare assays, for signal to noise a value of 10 is acceptable whereas Z factor is 0.4
Poor
What role does Z’ Factor play in a high-throughput screening campaign?
Used to exclude plates with poor quality, which indicates reliable and reproducible results.
What is the equation for Z’ Factor in high-throughput screening assays?
Z’ = 1 - 3 x (SD_positive control signal + SD_background)/ mean of both
How is the Coefficient of Variation (CV) for the positive control signal calculated?
CV_positive control signal = 100 x (SD_background / Mean_background).
What is the formula for calculating Signal-to-Noise ratio in HTS assays?
S/N = (Mean_positive control signal - Mean_background) / √[(SD_positive control signal)² + (SD_background)²].
Examples of assays, which is the best=1,2,3
1 is the best because the mean is the highest but has more variability.
Assay 2 and 3 have less variation and will be more precise
What are the 4 standard outcomes of HTS and explain.
TP,TN,FP,FN
Pan assays
What are the main classes of false positives?
Pan assays are compounds that contain functional groups that react with other components in the assay and give false positives.
Should remove these, false negatives are hard to find and accepted
Or compounds can interfere with the signal like luminescence inhibitors.
(l,r,pg,cc)
What are the common sources of error in HTS
Common sources of error include liquid handling equipment, robotic systems, plate geometry, and unintended changes in compound concentration.
Describe three cut-off criteria for hit selection
top %, % inhibition, compound outside 3xsd of control range
rep
How is reproducibility ensured in high-throughput screening?
Replicate analysis to distinguish between active compounds and non-reproducible compounds
