Biology Reference
In-Depth Information
vortex.cs.wayne.edu/ontoexpress/] [14]. Initial analysis used the non-filtered dataset,
that is, all differentially regulated probe sets against the full human oligonucleotide
geneset. We then looked at differentially expressed probes (
p
-value < 0.01) identi-
fied from our microarray analysis, and statistical significance values were calculated
for each category using the binomial test available in Onto-Express [15]. This makes
no assumptions about those probesets with good matches to known pig sequences.
However, only those probesets for which we could confidently assume orthology are
reported in the tables in this chapter. Here we present categories of gene ontology
based on a maximum pairwise
p
-value of 0.05 for the “biological processes.” To gain
a better understanding of the gene interactions (pathways) involved in the disease,
Pathway-Express was also applied to our data. In order to quantify the over/under
representation of each category, the library composition has been taken into account in
the presentation of the results.
Quantitative RNA Analyses Using Real-time PCR Methodology (qRT-PCR)
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR)
analysis using SYBR green and selected primers was carried out following the manu-
facturer's protocol (QIAGEN, QuantiTect SYBR Green RT-PCR) to confirm the mi-
croarray results. All probes and primers were designed using Express Primer 3 soft-
ware developed by the Whitehead Institute for Biomedical Research. The nucleotide
sequences of selected genes were obtained from GenBank, and the primer information
is shown in Table 1. The
PSMD2
(primers kindly provided by Ms. Gina Oliver and
Dr. Claire Quilter) was selected for use as the reference gene because it was previ-
ously shown to be a good control for pig brain (personal communication from Ms.
Gina Oliver and Dr. Claire Quilter) and was also shown to be one of the most constant
housekeeping genes in a human tissue study. The qRT-PCR was performed on 300
ng RNA equivalents in 25 μl/reaction/well on an Icycler (Bio-Rad Laboratories Ltd,
USA) (50°C for 60 min; 95°C for 15 min; 40 cycles of 95°C for 15 sec, 58°C for 30
sec, and 72°C for 30 sec). For each gene reactions were performed in triplicate to al-
low statistical evaluation of the data. The average Ct (threshold cycle) was used for
the analysis. Relative expression levels were calculated by using the 2
-(ΔΔCt)
method as
previously described [16].
Table 1.
Validation of array data by real-time PCR.
Microarray data
qRT-PCR data
Gene
name
Pig
homologene
Primer sequences
(5'-3')
Brain
(n-fold change)
Lung
(n-fold change)
Brain
(n-fold change)
Lung
(n-fold change)
PSMD2
Ssc. l642
F: tggggagaataagcgttttg
R: tattcatgaccccatgatgc
Ref
Ref
Ref
Ref
AKTI
Ssc.29760
F: tgggcgacttcatccttg
R: tggaagtggcagtgagca
ND
a
1.68
ND
2.19
CDC42
Ssc.6687
F: aaagtgggtgcctgagata
R: ctccacatacttgacagcc
-
b
2.03
-
7.38
LY96
Ssc.25550
F:cattgcacgaagagacataca
R: tgtattcacagtctctcccttc
1.37
3.32
6.91
9.23
