Biology Reference
In-Depth Information
Real-Time PCR master mix, 1.25
L of primers and probe
solution of TaqMan
®
Gene Ex Assays of the selected gene, and
RNase DNase free water in a total volume of 25
μ
L.
3. The reaction mixtures are loaded into optical 96-well plates,
sealed with the optical adhesive fi lm, and spun down for 1 min
at 4 °C. The reaction is performed in 7300 Real-Time PCR
system under the following conditions: 50 °C for 2 min and
95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and
60 °C for 1 min. Samples are amplifi ed in triplicate.
4. Results are expressed as a treated/reference sample ratio. The
comparative procedure applied is the Cq(2-
μ
Cq
) method
[
20
], that calculates changes in gene expression as a relative
fold change between the treated and the untreated (or calibra-
tor) sample. This procedure assumes that the amplifi cation
kinetics of the target and reference gene assays is equal.
Variation of the effi ciency of the reverse transcriptase as well of
the amount of the RNA used for reverse transcription can be
addressed by means the use of an endogenous control present
in each sample, such as a housekeeping gene like GAPDH or
the ribosomal protein L22 encoded by the RPL22 gene (
see
Note 5
).
ΔΔ
4
Notes
1. Before processing to the bioanalyzer, it is preferable to verify
the RNA integrity by agarose gel electrophoresis and to quan-
tify it by a spectrophotometer, considering that a pure RNA
has an absorbance ratio 260/280 of 1.8-2.0.
2. Unsupervised and supervised analyses (differential expressed
genes selection) can be performed using Partek Genomics
Suite using the ANOVA statistical implementation of this
package. Partek Genomics Suite can be used also to visualize
results using the agglomerative hierarchical clustering.
3. Pathway analysis can be supported by commercial and free-
available software such as Ingenuity Pathway Analysis (IPA),
Pathway studio, PathExpress, and Pathway miner among oth-
ers, which are platforms that provide genes/proteins relation-
ships as networks, pathways, or explore putative downstream
and upstream molecules, respectively, targeted or regulating,
the identifi ed pathways [
19
].
4. The best choice of a priming method (random hexamers, oli-
godT or gene-specifi c primers) for reverse transcription should
be determined experimentally. Sometimes, especially for long
transcripts, a combination of random primers and oligodT is
preferable.
