Transcript Noushin Farnoud Presentation

Genome-Wide RNAi Analysis
of
Growth and Viability in
Drosophila Cells
Michael Boutros, Amy A. Kiger, Susan Armknecht,
Kim Kerr, Marc Hild, Britta Koch, Stefan A. Haas,
Heidelberg Fly Array Consortium, Renato Paro,
Norbert Perrimon
Presented by: Noushin Farnoud
Goal
Whole Genome Sequence
Using RNAi to analyze LOF phenotypes
to characterize gene functions
List the putative Genes in cell growth
and viability
Functional Analysis of predicted
Genes
Cell viability assay:
Using Lucimnescent to correlate the
reduction of signal to dying cells
• Fluorometric method for estimating the number of viable
cells in culture by quantifying the amount of ATP present.
inhibitor of apoptosis :
positive control in screen
Fluorescence microscopy of Kc167
cells after 3 days treatment with RNAi
Z scores
• Indicates how far and in what direction an item
deviates from its distribution's mean.
• Especially useful when seeking to compare the
relative items from distributions with different
means and/or different standard deviations.
Method Reproducibility (5 days)
D-IAP1 phynotypes: positive control
Scatter plot of quantitative genomewide RNAi phenotypes
Correlation=0.86
Efficiency of the screen:
Negative control
Positive control
z = 2.9 +/- 0.8
• Averaged RNAi phenotypes of 72 genes
encoding all annotated ribosomal proteins were
tested.
Further analysis of essential gene
functions: Sequence-based
Classification of Genes
Quantitative Reproducibility
• Phenotypes for a highly represented
functional group of genes (nearly all of the
tested genes encoding ribosomal
components exhibited similar quantitative
phenotypes).
• Analysis of the phenotypes from
independent dsRNAs targeting the same
gene (show similar phenotypes).
Results of Quantitative analysis
• Identified many uncharacterized genes with
essential cellular roles (e.g, HDC14318 with
z=6.8 and 7.3).
• Identified uncharacterized genes with
phenotypes quantitatively similar to that of DIAP1 (z score 5). Examples: CG11700, a
ubiquitin-like gene, and CG15455, a gene
encoding an AML1-like transcription factor (z
scores 7.2 and 7.4 in Kc167 cells).
Classification of quantitative RNAi
phenotypes of selected genes
Flow cytometry analysis of propidium iodide
stained DNA after 3 days RNAi
Identification of anti-apoptotic gene
function by severity of RNAi viability
phenotypes
Results
• It suggests that the CG11700 gene may act in
the same pathway as D-IAP1 to directly prevent
Nc caspase-activated apoptotic cell death.
• In contrast, a set of essential transcription
factors may regulate complex responses for cell
fate, proliferation, and/or cell survival that
directly or indirectly initiate a partially caspasedependent apoptotic program.
Critiques
• Supplementary data !!!!!!
Scatter plot of quantitative genome-wide RNAi
phenotypes
“For example, D-IAP1
resulted in a severe
phenotype with z
scores of 6.1 and 7.0
in S2R and Kc167
cells, respectively”.
RNAi against ribosomal genes show
similar quantitative phenotypes
34 genes:
Similar quantitative
RNAi phenotype
putative falsenegative
RNAi experiments
Total number
Of annotated as
ribosomal
subunits
Lucimnescent cell viability assay
•
The assay determines the number of viable cells in culture by quantifying the amount
of ATP present - a marker of metabolically active cells.
The assay is designed for 96 or 384 well formats, making it ideal for automated high
throughput screening (HTS), of cell proliferation and cytotoxicity assays.
A homogeneous 'add, mix and measure' format makes the CellTiter-Glo assay simple
to use with no cell washing, media removal or multiple pipetting steps.
Measurements can be made on a CCD camera imaging device or a plate reading
luminometer (no injector is required) as little as 10 minutes after addition of the
reagent.
•
•
•
•
The CellTiter-Blue™ Cell Viability Assay is a homogeneous,
fluorometric method for estimating the number of viable
cells present in a cell population.
Similar to a video camera, digital camera use the lens which focuses the image onto
a Charge Coupled Device (CCD), which then converts the image into electrical
pulses. These pulses are then saved into memory.
In short, Just as the film in a conventional camera records an image when light hits it,
the CCD records the image electronically.
•
Z scores
• Indicates how far and in what direction an item deviates from its
distribution's mean.
• Especially useful when seeking to compare the relative standings of
items from distributions with different means and/or different
standard deviations.
• The number of standard deviations that a measurement is from the
mean. This quantity indicates the relative location of a measurement
within its distribution.