2. For free microRNA quantification, perform reverse transcription using TaqMan MicroRNA Reverse Transcription Kit and
miRNA-specific RT primers, following manufacturer’s instructions (see Note 18). Perform real-time quantitative PCR using
30 ng of obtained cDNA for each run. Prepare an RT-qPCR
mix containing cDNA, miRNA-specific PCR assay 20Â and
TaqMan Universal PCR Master Mix, no AmpErase UNG 2Â.
Run all RT-qPCR reactions, including no-template controls
and RT-minus controls, in duplicate, using the CFX96 Touch
Real-Time PCR Detection System. Calculate miR-221-3p relative expression using the comparative cycle threshold method
and use U6 snRNA (TM:001973) and let-7c-5p (TM:
000379) as reference since it remains constant in the assayed
samples by miR-profiling and quantitative RT-PCR analysis, as
previously reported [34, 46, 48]. A comprehensive discussion
focusing on the specificity of PNA-mediated miRNA inhibition
is presented in Note 19. Additional information on required
controls for validation of the PNA-mediated alteration of
biological functions is presented in Notes 20 and 21.
3.6 Studies on Cell
Viability
1. Evaluate cell viability after 72 h contact with argininocalix[4]
arene 1 using Muse Count and Viability Kit and Muse Cell
Analyzer. Perform analysis, using 50 μL of suspension cells
(obtained after U251 trypsinization) added with 225 μL of
Muse Count and Viability Reagent. Incubate the mixture at
room temperature for 5 min, protected from the light and then
acquire 1 Â 10
3 events using Muse Cell Analyzer. Evaluate the
effects of argininocalix[4]arene 1 on cell growth determining
the cell number/mL using a Z2 Coulter Counter. Examples
focusing on the possible cytotoxicity of argininocalix[4]arene
1 are presented in Note 22.
4 Notes
1. PNAs are usually designed according to the following criteria:
(a) length of at least 18 bases; 18 bases ensures high stability of
the PNA:RNA duplex, and suitable efficiency of the synthesis
on large scale (this issue is particularly important if ex vivo and
in vivo studies are programmed, requiring large quantity of
PNAs), whereas longer sequences might prove difficult to
obtain with proper yields and purities; (b) lack of selfcomplementarity both in the antiparallel and parallel orientation since the PNA:PNA duplex is more stable than PNA:DNA
and PNA:RNA complexes; (c) minimal length of complementary sequences when the complete set of the transcriptome is
considered, as evaluated by bioinformatic analyses (such as a
BLAST search); after this estimation, it is also important to
PNA Delivery with Argininocalix[4]arene
133
miRNA-specific RT primers, following manufacturer’s instructions (see Note 18). Perform real-time quantitative PCR using
30 ng of obtained cDNA for each run. Prepare an RT-qPCR
mix containing cDNA, miRNA-specific PCR assay 20Â and
TaqMan Universal PCR Master Mix, no AmpErase UNG 2Â.
Run all RT-qPCR reactions, including no-template controls
and RT-minus controls, in duplicate, using the CFX96 Touch
Real-Time PCR Detection System. Calculate miR-221-3p relative expression using the comparative cycle threshold method
and use U6 snRNA (TM:001973) and let-7c-5p (TM:
000379) as reference since it remains constant in the assayed
samples by miR-profiling and quantitative RT-PCR analysis, as
previously reported [34, 46, 48]. A comprehensive discussion
focusing on the specificity of PNA-mediated miRNA inhibition
is presented in Note 19. Additional information on required
controls for validation of the PNA-mediated alteration of
biological functions is presented in Notes 20 and 21.
3.6 Studies on Cell
Viability
1. Evaluate cell viability after 72 h contact with argininocalix[4]
arene 1 using Muse Count and Viability Kit and Muse Cell
Analyzer. Perform analysis, using 50 μL of suspension cells
(obtained after U251 trypsinization) added with 225 μL of
Muse Count and Viability Reagent. Incubate the mixture at
room temperature for 5 min, protected from the light and then
acquire 1 Â 10
3 events using Muse Cell Analyzer. Evaluate the
effects of argininocalix[4]arene 1 on cell growth determining
the cell number/mL using a Z2 Coulter Counter. Examples
focusing on the possible cytotoxicity of argininocalix[4]arene
1 are presented in Note 22.
4 Notes
1. PNAs are usually designed according to the following criteria:
(a) length of at least 18 bases; 18 bases ensures high stability of
the PNA:RNA duplex, and suitable efficiency of the synthesis
on large scale (this issue is particularly important if ex vivo and
in vivo studies are programmed, requiring large quantity of
PNAs), whereas longer sequences might prove difficult to
obtain with proper yields and purities; (b) lack of selfcomplementarity both in the antiparallel and parallel orientation since the PNA:PNA duplex is more stable than PNA:DNA
and PNA:RNA complexes; (c) minimal length of complementary sequences when the complete set of the transcriptome is
considered, as evaluated by bioinformatic analyses (such as a
BLAST search); after this estimation, it is also important to
PNA Delivery with Argininocalix[4]arene
133