Biology Reference
In-Depth Information
Fig.
3
Effects of 48 h treatment of K562 (
a
), IB3-1 (
b
), MCF-7 (
c
), and MDA-MB-231 (
d
) cells with 2
M PNA-
a221 and Rpep-PNA-a221 on hybridization signal of miR-221. (
e
) Accumulation of miR-221, miR-let-7c and
miR200c in MDA-MB-231 cells treated for 48 h with 2
μ
M PNA-a221 and Rpep-PNA-a221. RT-PCR was done
as described in Subheadings
2
and
3
and in Brognara et al. [
22
]
μ
Fig.
4
Accumulation of p27
Kip1
mRNA (
a
) in MDA-MB-231 cells treated for 96 h with 2
M PNA-a221 and Rpep-
PNA-a221. (
b
) Western blotting performed on the same cellular populations using antibody against p27
Kip1
and
against
μ
β
-actin as reference protein. Modifi ed from Brognara et al. [
22
]
3.4 Studies on
Alteration of Gene
Expression
1. For gene expression analysis (
see
Note 6
) reverse transcribe
500 ng of the total RNA by using random hexamers. Carry out
quantitative real-time PCR assays using gene-specifi c double
fl uorescently labeled probes. The relative expression is calcu-
lated using the comparative cycle threshold method and, as ref-
erence genes, the endogenous control human 18S rRNA [
21
].
2. RT-qPCR analyses. The effects on p27
Kip1
mRNA, shown in
Fig.
4a
, indicate that no change of p27
Kip1
mRNA content
occurs in MDA-MB-231 cells in the presence of PNA-a221,
whereas signifi cant increase of p27
Kip1
mRNA is observed with
the Rpep-PNA-a221 (
p
< 0.05).
