Biology Reference
In-Depth Information
by a
T
-test with a
p
-value
≤
0.05 and a fold change
≥
+1.5 and
−1.5, and then iteratively testing the enrichment of each
term annotation. Alternately, all genes from a microarray
experiments are uploaded without any pre-selection of signifi -
cant genes, in order to allow also minimal transcription changes
contributing to the enrichment analysis.
3. Besides the classifi cation of genes based on Gene Ontology,
the pathway discovery is another important downstream inves-
tigation method, useful to extrapolate biological results.
Pathway analysis treats genes in the same pathway as a gene set
(
see
Note 3
). The advantage of analyzing genes as a set is that
it can detect coordinate changes that are usually moderate or
weak at single gene level.
4. The analysis of differentially expressed genes and related path-
ways in a given experimental condition can be complemented
with insights of putative regulators elements such as transcrip-
tion factors and microRNAs, based on promoter binding sites
and regulative mRNA sequences identifi ed in the co-expressed
genes. Some common tools for transcription factor binding
site or microRNA prediction and underlying algorithms are
Ingenuity Pathway Analysis, oPOSSUM, TFSCAN, and
TFSEARCH [
19
].
≤
1. Retro-transcription reagents are stored at −20 °C and thawed
before use. Add 300-500 ng of purifi ed RNA to the nuclease-
free microcentrifuge tube along with the following compo-
nents: 250 ng of random hexamers, 1
3.10 cDNA Synthesis
for Microarray
Genes Validation
by Real-Time PCR
μ
L 10 mM dNTP mix,
L (
see
Note 4
).
2. Heat mixture at 65 °C for 5 min and incubate on ice for at least
1 min.
3. After a brief centrifugation, add 7
and DEPC-treated water up to 13
μ
μ
L/tube of a solution
composed of 1
μ
L 0.1 M DTT, 4
μ
L 5× First-Strand Buffer,
1
μ
L of RNase Inhibitor, 1
μ
L of reverse transcriptase and mix
by pipetting.
4. Samples are incubated at 25 °C for 5 min.
5. Next, samples are incubated at 50 °C 50 min.
6. The reaction is inactivated at 70 °C 15 min. The cDNA can
now be used as template for amplifi cation by Real-Time PCR.
3.11 Microarray
Genes Validation
by Real-Time PCR
1. Gene expression changes in the treated samples are measured
by relative quantifi cation with respect to the untransduced
reference sample. An example of the changes in gene expres-
sion induced by three different viral vectors is shown in Fig.
4
.
2. Use 1/20 of retro-transcribed cDNA for amplifi cation of the
target gene in a solution composed of 12.5
μ
L of TaqMan
®
