Biology Reference
In-Depth Information
fi cation, or detection of viral antigen-positive tissues were used to confi rm the organic
infection by PRV, and diseases including swine fever (SF), Porcine Reproductive, and
Respiratory Syndrome Virus (PRRSV) and other potential bacterial infections which
could be clinically and pathologically confused with PRV infection were excluded by
viral antigen, antibody identifi cation, and polymerase chain reaction (PCR) detection.
Six piglets aged from 2 to 4 days (commercial breed Landrace X Yorkshire) which
were infected by PRV but not by the other tested diseases (see above) and three healthy
piglets (not infected, and negative for all tests under the strict criteria used above),
matched for age and breed from the same farm were used in this experiment. All ex-
periments were carried out in strict accordance with accepted HuaZhong Agricultural
University, China, and governmental policies.
Microarray Experimental Design
Total mRNA samples from the brains and lungs of the three normal piglets were pooled
for the reference mRNA. Ten independent RNA samples (six biological replicates for
brain and four biological replicates of lung) from the six infected piglets were paired
with the reference sample for hybridization on two-color microarrays. Using a dye-
swap configuration, comparing each sample provides technical replicates to adjust for
dye bias [9]. A total of 20 slides were used in this study.
RNA Purification
Total mRNA was prepared using Qiazol reagent (Qiagen, Crawley, West Sussex, UK)
following the manufacturer's instructions. A second purification step was performed
immediately post extraction on the isolated total mRNA using the RNeasy Midi kit
(Qiagen Inc., Valencia, CA) and each sample was treated with DNase (20 U of grade
I DNase; Roche, Lewes, UK) to remove any genomic contamination following the
manufacturer's instructions. With a cut-off of 150 bp, 5S rRNA, and tRNAs were re-
moved from the samples by the columns, limiting interference in downstream experi-
ments. The RNA concentration and integrity were assessed on the Nanodrop ND-1000
spectrophotometer (Nanodrop, USA) and on the Agilent 2,100 bioanalyzer system
(Agilent Technologies, Palo Alto, CA), using an RNA 6000 Nano LabChip kit.
SMART Amplification and Labeling of the Samples
The extracted RNA was amplified using the SMART amplification protocol (BD Smart
TM Amplification Kit, UK) and labeled with Cy5 or Cy3 using Klenow enzyme as
described by Petalidis et al. 2003 [10] with two modifications; (a) a constant number
of 14 cycles was used, and (b) for the labeling step, 1 μl of Cy3 or Cy5-dCTP was used
with 22 μl (250 ng) of second strand cDNA. The labeled products were purified using
G50 columns, according to manufacturer's instructions (Amersham Biosciences, UK).
Labeled samples were combined and precipitated for at least 2 hr at 20°C with 2 μl of
human Cot-1 DNA, 1 μl PolyA (8 μg/μl), 1 μl yeast tRNA (4 μg/μl), 10 μl Na acetate
(3 M, pH 5.2), and 250 μl 100% ethanol.
Microarray Hybridization and Scanning
The labeled product was re-suspended in 40 μl hybridization buffer (40% deionised
formamide, 5 × SSC, 5 × Denhart's, 1 mM Na Pyrophosphate, 50 mM Tris pH 7.4 and
