MIAME COMPLIANCE CHECKLIST:
Moser, Freitas, Arasu, Gibson
Gene expression profiles associated with the transition to parasitism in Ancylostoma
caninum larvae
Experiment Design
Type of experiment:
Expression analysis of A. caninum genes in relation
to the presence of canine serum, mimicking the
entry into a host and the resumption of a parasitic
lifestyle.
Experimental factors:
Two A. caninum strains: 1 Maryland. 1 Wake
County, North Carolina
Two treatments: Arrested, infective third stage
A. caninum larvae. Serum-stimulated third stage A.
caninum larvae
Number of hybridizations:
24
Type of Reference:
NONE
Hybridization Design:
Complete loop with 12 replicates: see paper for
experiment design, and headers in raw data file
Quality Control:
Each of the 4 samples (2 treatments by 2 strains)
consisted of >100,000 larvae, so no individual
variance was measured.
Supplementary Data URL:
http://statgen.ncsu.edu/ggibson/SupplInfo/SupplInfo10.htm
Samples used, Extract preparation and labeling:
Biological Samples:
Ancylostoma caninum (canine hookworm). Third
stage arrested, infective larvae (iL3) grown in
culture from eggs collected from laboratoryinfected beagles. Pre-parasitic A. caninum iL3
larvae were collected from charcoal cultures of egginfected feces of laboratory Beagle dogs infected
with each of the two laboratory strains of
hookworm. These strains were derived from natural
populations of parasitized dogs in Maryland (kindly
provided by Dr. Thomas Nolan, University of
Pennsylvania) and Wake County, North Carolina.
Harvested larvae were washed several times in
medicated (20 mg/ml gentamycin and lincomycin)
phosphate-buffered saline (PBS).
Biological manipulations:
To obtain serum stimulated feeding ssL3 larvae,
iL3 were incubated in PBS (5000 L3/500 μL) at
37ºC/5% CO2 for 20-24 hrs in the presence of 5%
normal dog serum in 24 well plates. To check for
feeding and reactivation status, about 250 larvae in
100 l PBS were incubated at 37º C/5% CO2 for 2
hours in the presence of an equal volume of a
5mg/mL fluorescein isothiocyanate-conjugated
bovine serum albumin (FITC-BSA; Sigma)
solution. The larvae were repeatedly washed in
PBS before examination by fluorescent microscopy.
At least 50 larvae from each well were scored;
positive feeding larvae display fluorescent intestinal
tracts, and a batch of worms was considered
‘serum-stimulated’ if greater than 85% of the larvae
were positive.
Larvae were pooled according to treatment (iL3 or
ssL3) and flash-frozen prior to RNA extraction.
Total RNA was extracted using RNAstat-60 reagent
(Tel-Test) manufacturer’s protocol.
One round of experiments (12 hybridizations
representing a complete block of the 2 treatments by
2 strains) was completed using 20 ug total RNA for
each channel of each hybridization.
One round of the experiment was completed using
linear amplified RNA from the Agilent Low Input
Fluorescent Liner Amplification Kit (5184-3523).
Briefly, total RNA was aliquoted into 500ng
amounts, which were amplified following
manufacturer’s instructions, resulting in 1.5 to 45
ug of acRNA.
Labeling protocols:
Direct labeling: Total RNA was reverse transcribed
with SuperScript II (Gibco) in the presence of Cy3dUTP or Cy5- dUTP (Agilent).
Indirect labeling: 3 ug of acRNA was fluorescently
labeled with Cy3 or Cy5 following the aminoallyl
indirect labeling protocol from The Institute for
Genomic Research SOP#M004 found at
http://pga.tigr.org/sop/M004_1a.pdf
External Controls:
None
Hybridization Procedures and Parameters:
Hybridization Protocol:
Hybridizations were performed using printed slides
treated with 5X SSC, 0.1% SDS, and 1% BSA for
45 minutes to block non-specific binding.
Hybridization reactions containing labeled cDNA,
5xSSC, 1ug poly-adenine oligo, 5X Denhardt’s
solution, 50% formamide, 0.5% SDS, and calf
thymus DNA were placed onto the prepared slides
and incubated for 16- 20 hours at 42ºC. All
hybridizations were performed between March 25th,
2003 and Jan 22nd, 2004.
Washing Protocol:
After overnight hybridization, the slides were
washed with a series of 3 high stringency washes of
1 each of 1xSSC/0.2%SDS, 0.1xSSC/0.2%SDS,
and three washes with 0.1xSSC.
Measurement Data and specifications:
Scanning and software:
ScanArray 4000 Microarray Analysis System
Scanner (Packard Bioscience).
48 aquired TIFF images were analyzed using
ScanAlyze software version 2.50 which is freely
available at http://rana.lbl.gov/Eisensoftware.htm
Type of Data:
Raw fluorescent intensity values (NOT background
subtracted).
Log base 2 tranformations of RFI values. Spots
with log2 RFI values at or below background levels
were removed from further analysis (Raw value
202.81, log2 value 7.664).
Data Files:
48 raw data files in Microsoft Excel format were
combined into 1 JMP v. 5 sheet containing the log2
RFI values for each spot along with identifying
information from each of the 48 channels (2
channels per hybridization).
JMP file imported into SAS v. 8.2.
Data transformation:
Took the average log2 RFI values for each spot
across all 48 channels, and removed spots with
average value at or below background levels.
2-step Mixed Model Analysis of Variance: SAS
code freely available at
http://statgen.ncsu.edu/ggibson/Manual.htm
-Compute RFI by removing overall dye, array, and
batch effects
-Evaluate treatment specific effects, strain specific
effects, and the treatment*strain interaction effects.
Volcano plots were used to evaluate magnitude and
significance of the relative expression differences
between the two treatments and between the two
strains using the DIFFS output from the SAS code.
Two-way hierarchical clustering was evaluated in
JMP.
Final Gene Expression Data:
Worksheet 1: Selected Gene list with annotations
Worksheet 2: Results of Mixed Model ANOVA
Worksheet 3: Raw data file
Array Design:
cDNA arrays printed in duplicate with a pin and
loop arrayer onto amino-silane coated slides
(Corning GAPs II).
UV crosslinking binds DNA to slide.
Slides stored in desk-top desiccator until needed.