MIAME COMPLIANCE CHECKLIST:
Passador-Gurgel, Hsieh, Hunt, Deighton and Gibson (2006)
Quantitative Trait Transcripts for Nicotine Resistance in Drosophila melanogaster
Hsieh, Passador-Gurgel, and Gibson (2006)
Mixture Modeling of Transcriptional Population Structure in Drosophila melanogaster
Experiment Design
Type of Experiment:
Quantitative analysis of response to nicotine in lines from
different populations of Drosophila melanogaster
Experimental Factors:
Two populations: California (50 lines) and North
Carolina (58 lines) female fruit flies
Two treatments: control and nicotine
Number of Hybridizations:
332
Type of Reference:
None
Hybridization Design:
Three complete loops, one for each population
(California, North Carolina using total RNA and
North Carolina using amplified RNA). For each
loop, each line by treatment was compared to a
different line by treatment.
Quality Control:
Each of the 108 samples (50 California lines and 58
North Carolina lines in control or nicotine food)
with at least two RNA amplifications was pooled.
Each RNA sample was split into two separate cDNA
synthesis reactions; these were then pooled and
re-split into two separate tubes (one for each
labeling reaction).
Each comparison of samples was balanced for the two
dye types.
Supplementary Data URL:
http://statgen.ncsu.edu/ggibson/SupplInfo/SupplInfo12.htm
Samples used, Extract preparation and labeling:
Biological Samples:
Drosophila melanogaster (fruit fly)
108 inbread lines: 58 lines from a North Carolina
population and 50 lines from a California
population
Heads from female flies from each line by treatment were
used for RNA extraction
Heads were isolated by sieving whole flies that had been
frozen in liquid nitrogen through a fine gauze
(710μm) onto Whatman filter paper and stored at
-80C
Biological Manipulations:
RNA was extracted using TRIzol manufactures protocol
(Invitrogen), and stored at –80C
RNA was linear amplified using Agilent Low Input
Fluorescent Linear Amplification Kit (5184-3523)
*Protocol was modified by only performing a
single round of amplification to get amplified
complementary RNA
Labeling Protocols:
Labeled total or amplified RNA samples with Cy3 or Cy5
using aminoallyl indirect labeling protocol from
The Institute for Genomic Research (SOP#M004)
External Controls:
None
Hybridization procedures and parameters:
Hybridization protocol:
Hybridizations were performed for 20 hours at 42C.
Washing Protocol:
Washings were performed using a standard series of three
high stringency washes
Measurement data and specifications:
Scanning and software:
ScanArray 4000 Microarray Analysis System Scanner
(Packard Bioscience).
Six hundred sixty four (two for each array) acquired TIFF
images were analyzed using UCSF Spot software
(http://www.jainlab.org/downloads.html)
Data Files:
332 raw data files in text format
3 data files for SAS import with log2 values for each
array, with spots with high background noise and
poor PCR amplification removed
Type of Data:
Raw fluorescence intensity values with background
values subtracted
Log base 2 transformations of raw subtracted
fluorescence values
Data Transformation:
Subtracted background average from the spot raw
intensity levels for each dye channel
2 -Step Mixed Model ANOVA:
-Step 1: Compute relative fluorescence intensities
(RFI), by removing overall dye and array
effects
-Step 2: Evaluate treatment specific effects, line
specific effects, population specific effects,
treatment and population interaction effects
and line and treatment specific effects.
Volcano plots were used to evaluate the significance of
expression against the magnitude of the
expression (plotted as negative log p-value against
log base 2 values).
Correlation analyses between control- or nicotine-induced
expression and nicotine survival time were carried
out using regression analyses.
Final Gene Expression Data: http://statgen.ncsu.edu/ggibson/SupplInfo/SupplInfo12.htm
http://statgen.ncsu.edu/ggibson/SupplInfo/SupplInfo13.htm
Worksheet 1: Gene list with annotations
Worksheet 2: Raw Data file
Worksheet 3: Estimates of LSmeans from Mixed Model ANOVA
Worksheet 4: Estimates of Differential Expression by Drug
Worksheet 5: Estimates of Differential Expression by Population
Array Design:
General:
cDNA arrays printed with a pin and loop printer on
amino propyl silane coated slides (Corning Gaps II)
UV Crosslinking binds DNA to the slide