Molecular Methods for Validation of the Biological Activity of Peptide Nucleic Acids Targeting MicroRNAs - miRNA Maturation: Methods and Protocols - page 161

2 Materials

2.1 Peptide Nucleic
Acids (PNAs)

PNAs can be purchased from several companies, including
Panagene, Inc. ( www.panagene.com ) . Alternatively, PNAs against
miRNAs can be synthesized following the procedures described in
Manicardi et al. [ 2 ] and Fabbri et al. [ 3 ]. The data here presented
are based on PNAs described in the paper by Brognara et al. [ 22 ].
The sequences of PNA-a221 and Rpep-PNA-a221 are reported in
Table 1 ; fl uorescein-labeled PNAs were used for the uptake
experiments.

2.2 Cell Culture

1. RPMI-1640 medium supplemented with 10 % fetal bovine
serum, 50 units/ml penicillin, and 50 mg/ml streptomycin.
2. LHC-8 basal medium without gentamycin.
3. D-MEM medium.
4. 2 mM L -Glutamine.
5. Human erythroleukemic K562 cells.
6. Bronchial epithelial IB3-1 cell line, derived from Cystic Fibrosis
patients.
7. Human breast cancer cell lines MCF-7 and MDA-MB-231.
8. To determine the effects on proliferation, cell growth is moni-
tored using a Z1 Coulter Counter (Coulter Electronics,
Hialeah, FL, USA).
9. Cell incubation and monitoring system like BioStation instru-
ment (BioStationIM, Nikon Instruments).

2.3 FACS Analysis

1. Trypsin-EDTA.
2. Fetal bovine serum, FBS.
3. Phosphate-buffered saline, PBS.
4. Determination of cellular uptake: fl uorescence intensity was
detected using the FACScan (Becton Dickinson, Franklin
Lakes, NJ, USA). Cells were analyzed using the CellQuest™
version 3.3 software (Becton Dickinson, Franklin Lakes,
NJ, USA).

Table 1
Sequences of PNAs

PNA-a221 H-AAACCCAGCAGACAATGT-NH 2

Fl-PNA-a221 Fl-AEEA-AAACCCAGCAGACAATGT-NH 2

Rpep-PNA-a221 H-RRRRRRRR-AAACCCAGCAGACAATGT-NH 2

Fl-Rpep-PNA-a221 Fl-AEEA-RRRRRRRR-AAACCCAGCAGAC
AATGT-NH 2
Fl fl uorescein; AEEA 2-(2-aminoethoxy)ethoxyacetyl spacer