Biology Reference
In-Depth Information
Fig.
2
FACS analysis performed after 24 h of culturing K562 (
a
), IB3-1 (
b
), MCF-7 (
c
), and MDA-MB-231
(
d
) cells in the absence (ctrl) or in the presence of 2
μ
M fl uorescein labeled Fl-PNA-a221 (
dashed arrows
) and
Fl-Rpep-PNA-a221 (
dotted arrows
) molecules
Transcription Kit; perform real-time PCR according to the
manufacturer's protocols. Use 20 ng per sample for the assays.
Perform all RT reactions, including no-template controls and
RT-minus controls, in duplicate using the 7700 Sequence
Detection System version 1.7. Calculate the relative expression
using the comparative cycle threshold method and use U6
snRNA as reference to normalize all RNA samples, since it
remains constant in the assayed samples by miR-profi ling and
quantitative RT-PCR analysis, as reported previously [
3
,
21
].
3. When K562, IB3-1, MCF-7, and MDA-MB-231 cells are cul-
tured in the presence of PNA-a221 and Rpep-PNA-a221 simi-
lar effects were observed on miRNA-221. After RNA isolation,
perform RT-qPCR following protocols like described in the
chapter by Zoellner et al. and apply to analysis of miR-221 [
3
].
Figure
3a-d
demonstrate that the miR-221-specifi c hybridiza-
tion signal is strongly reduced only when RNA is isolated from
cells cultured for 48 h in the presence of Rpep-PNA-a221,
while no major effects are observed for PNA-a221 (
see
Note 5
).
4. Figure
3e
shows that these effects are restricted to miR-221,
since, despite the fact that some alteration of miRNA content
occurs, no suppression of accumulation of miR-200c and miR-
let-7c has been obtained. These data demonstrate specifi city of
PNA-mediated effects.
