6. Check the purity of the final compound by ESI-MS and
1 H
NMR. It is possible to compare the collected spectra with the
characterization data of the compound as reported [61].
3.2 Production of
Argininocalix[4]
arene 1/PNA
Complexes
1. Resuspend argininocalix[4]arene 1 in a solution of EtOH/
H 2 O/DMSO (2/2/1 v/v) under sterile condition.
2. Prepare a transfection mixture constituted by RPMI-1640
medium added with argininocalix[4]arene 1 at final concentration of 2.5 μM and 2 μM (final concentration) of appropriate
PNA (employed PNAs are listed in Subheading 2.2).
3. Incubate the mixture for 20 min at room temperature, without
serum. After the incubation, add 10% (v/v) of FBS.
4. Remove cell culture medium and replace it with the transfection mixture. Maintain the transfection mixture in contact with
cells until the end of the treatment.
3.3 Human Cell
Lines, Culture
Conditions, and
Treatment with PNA
and Argininocalix[4]
arene 1
1. Culture U251 cells [64] in humidified atmosphere of 5% CO 2 /
air in RPMI-1640 medium supplemented with 10% FBS, penicillin/streptomycin. The experiments aimed at determining
the working concentrations of calix[4]arenes are discussed in
Note 14.
2. Add to the cell culture the argininocalix[4]arene 1/PNA-a221
complex (equivalent to 2.5 μM of argininocalix[4]arene 1 and
2 μM of PNA-a221) to evaluate PNA-a221 uptake and
PNA-a221 pro-apoptotic effects. Use 2 μM R8-PNA-a221 as
transfection positive control (this PNA is able to be transfected
without vector employment). Monitor cell growth, according
to cell number/ml, usually, after 72 h of treatment, using the
Z2 Coulter Counter. The comparison of the effects of antimiRNA PNAs with those of other anti-miRNA molecules is
presented in Note 15.
3.4 PNAs Cellular
Uptake
1. Detach cells by trypsinization, wash them twice with DPBS 1Â,
and suspend in 150 μL of DPBS 1Â. Analyze cell suspension by
FACS analysis, using FACScan, and verify cells for FITC fluorescence. For each sample acquire 30,000 events and analyze
data using CellQuest Pro Software. Expected results of this
methodology are briefly discussed in Notes 16 and 17.
3.5 RNA Extraction
and Real-Time
Quantitative Analysis
of Free miRNAs
1. Trypsinize cells and collect them by centrifugation at 200 Â g
for 6 min at 4
C, wash with PBS, and lyse with 1 mL of
Tri-Reagent, according to manufacturer’s instructions. Wash
the isolated RNA once with cold 75% ethanol, dry and dissolve
in an appropriate volume of nuclease-free water. Store the
obtained RNA at À80
C until the use.
132
Alessia Finotti et al.
1 H
NMR. It is possible to compare the collected spectra with the
characterization data of the compound as reported [61].
3.2 Production of
Argininocalix[4]
arene 1/PNA
Complexes
1. Resuspend argininocalix[4]arene 1 in a solution of EtOH/
H 2 O/DMSO (2/2/1 v/v) under sterile condition.
2. Prepare a transfection mixture constituted by RPMI-1640
medium added with argininocalix[4]arene 1 at final concentration of 2.5 μM and 2 μM (final concentration) of appropriate
PNA (employed PNAs are listed in Subheading 2.2).
3. Incubate the mixture for 20 min at room temperature, without
serum. After the incubation, add 10% (v/v) of FBS.
4. Remove cell culture medium and replace it with the transfection mixture. Maintain the transfection mixture in contact with
cells until the end of the treatment.
3.3 Human Cell
Lines, Culture
Conditions, and
Treatment with PNA
and Argininocalix[4]
arene 1
1. Culture U251 cells [64] in humidified atmosphere of 5% CO 2 /
air in RPMI-1640 medium supplemented with 10% FBS, penicillin/streptomycin. The experiments aimed at determining
the working concentrations of calix[4]arenes are discussed in
Note 14.
2. Add to the cell culture the argininocalix[4]arene 1/PNA-a221
complex (equivalent to 2.5 μM of argininocalix[4]arene 1 and
2 μM of PNA-a221) to evaluate PNA-a221 uptake and
PNA-a221 pro-apoptotic effects. Use 2 μM R8-PNA-a221 as
transfection positive control (this PNA is able to be transfected
without vector employment). Monitor cell growth, according
to cell number/ml, usually, after 72 h of treatment, using the
Z2 Coulter Counter. The comparison of the effects of antimiRNA PNAs with those of other anti-miRNA molecules is
presented in Note 15.
3.4 PNAs Cellular
Uptake
1. Detach cells by trypsinization, wash them twice with DPBS 1Â,
and suspend in 150 μL of DPBS 1Â. Analyze cell suspension by
FACS analysis, using FACScan, and verify cells for FITC fluorescence. For each sample acquire 30,000 events and analyze
data using CellQuest Pro Software. Expected results of this
methodology are briefly discussed in Notes 16 and 17.
3.5 RNA Extraction
and Real-Time
Quantitative Analysis
of Free miRNAs
1. Trypsinize cells and collect them by centrifugation at 200 Â g
for 6 min at 4
C, wash with PBS, and lyse with 1 mL of
Tri-Reagent, according to manufacturer’s instructions. Wash
the isolated RNA once with cold 75% ethanol, dry and dissolve
in an appropriate volume of nuclease-free water. Store the
obtained RNA at À80
C until the use.
132
Alessia Finotti et al.