Molecular Methods for Validation of the Biological Activity of Peptide Nucleic Acids Targeting MicroRNAs - miRNA Maturation: Methods and Protocols - page 162

2.4 RNA Extraction

1. Trypsin-EDTA.
2. Fetal bovine serum, FBS.
3. Phosphate-buffered saline, PBS.
4. Tri-Reagent TM , for cell lysis.

2.5 Real-Time
Quantitative PCR
of MicroRNA

1. Primers and probes can been obtained from local supplier, like
Applied Biosystems.
2. Reverse transcriptase (RT) reactions are performed using the
TaqMan ® MicroRNA Reverse Transcription Kit (Applied
Biosystems, Foster City, CA, USA).
3. Real-time PCR machine, like the 7700 Sequence Detection
System version 1.7 (Applied Biosystems, Foster City, CA,
USA).

1. Primers and probes to assay p27 Kip1 (Assay ID: Hs00153277.
m1) and the endogenous control human 18S rRNA can be
purchased from Applied Biosystems (Applied Biosystems,
Foster City, CA, USA).
2. Reverse transcriptase (RT) reactions are preferentially per-
formed using the TaqMan ® Reverse Transcription Kit (Applied
Biosystems, Foster City, CA, USA). RNAs are reverse tran-
scribed by using random hexamers.

2.6 Taqman RT-qPCR
for Gene Expression
Analysis

1. SDS-denaturing buffer (1×): 62,5 mM Tris-HCl (pH 6.8),
2 % SDS, 50 mM DTT, 0.01 % bromophenol blue, 10 % glyc-
erol for cytoplasmic extracts.
2. SDS-PAGE running buffer: 25 mM Tris-HCl, pH 8.3,
192 mM glycine, 0.1 % SDS.
3. Biotinylated protein ladder (size range of 9-200 kDa).
4. Nitrocellulose membrane: 20

2.7 Western Blotting

m nitrocellulose membrane.
5. Western blot transfer buffer: 25 mM Tris, 192 mM Glycine,
5 % methanol.
6. Pre-staining Solution: Ponceau S Solution.
7. Tris-buffered saline, TBS: 10 mM Tris-HCl (pH 7.4), 150 mM
NaCl.
8. TBS containing 0.1 % Tween-20 (TBS/T).
9. Blocking solution: 5 % nonfat milk in TBS/T buffer. Store at
4 °C.
10. Primary monoclonal antibodies.
11. HRP-conjugated secondary antibody.
12. Chemoluminescent substrate like LumiGLO ® (0.5 ml 20×
LumiGLO ® , 0.5 ml 20× Peroxide, and 9.0 ml Milli-Q water),
Cell Signaling.

μ