3. The intracellular miR-29 and its target Mcl-1 amount is
measured post nano-electroporation using real-time PCR (see
Note 6).
4. It is observed that the increased miR-29b could significantly
suppress the Mcl-1 expression in living cells which necessitates
the study of dose dependent manner in miR-29- based gene
therapy (Fig. 2c).
5. Total RNA extraction from miR-29b transfected cells is carried
out by using Trizol and cDNA is made using random primers.
6. Real-time PCR is performed by using TaqMan gene expression
assay probe primer sets for miR-29b and Mcl-1 by ViiA 7 RealTime PCR System.
7. The expression of target genes relative to internal control gene
is calculated using the threshold cycle number (C t ). The relative target gene expression for each condition is normalized to
control and fold change determined using the comparative
method (2
(-ΔΔC t ) ).
8. After the transfection of miR-29b, the cardiomyocytes are
stained with JC-1 to determine the mitochondrial membrane
potential (see Note 7).
9. The stained cardiomyocytes are rinsed twice with media and
analyzed with a flow cytometry.
10. As the silicon chip is nontransparent, imaging the on-chip cell
trapping and FAM-ODN delivery is performed on an upright
microscope (Leica DM2500).
11. An inverted microscope (Nikon Eclipse Ti) is used to record
fluorescent expressions of cells cultured off-chip.
12. Two-sided student T-test is used to determine the significance
for data with Gaussian distribution and equal variances. Groups
with p-values <0.05 are considered significant difference.
4 Notes
1. As the NK cells are suspension cells, dielectrophoresis (DEP)
force is applied to trap cells on the nanochannel before run
nanoelectroporation. Details of DEP for cell capture are
provided in [13]. To maximize ▽E
2 on the NEP device and
to minimize the slew rate of the circuit, the operational amplifiers in the circuit are configured for open loop operation, and a
square wave feed is selected over other functions.
2. For a successful cell electroporation, the condition of threshold
voltage greater than resting transmembrane potential should
be met.
3D Nanochannel Electroporation for Macromolecular Nucleotide Delivery
75
measured post nano-electroporation using real-time PCR (see
Note 6).
4. It is observed that the increased miR-29b could significantly
suppress the Mcl-1 expression in living cells which necessitates
the study of dose dependent manner in miR-29- based gene
therapy (Fig. 2c).
5. Total RNA extraction from miR-29b transfected cells is carried
out by using Trizol and cDNA is made using random primers.
6. Real-time PCR is performed by using TaqMan gene expression
assay probe primer sets for miR-29b and Mcl-1 by ViiA 7 RealTime PCR System.
7. The expression of target genes relative to internal control gene
is calculated using the threshold cycle number (C t ). The relative target gene expression for each condition is normalized to
control and fold change determined using the comparative
method (2
(-ΔΔC t ) ).
8. After the transfection of miR-29b, the cardiomyocytes are
stained with JC-1 to determine the mitochondrial membrane
potential (see Note 7).
9. The stained cardiomyocytes are rinsed twice with media and
analyzed with a flow cytometry.
10. As the silicon chip is nontransparent, imaging the on-chip cell
trapping and FAM-ODN delivery is performed on an upright
microscope (Leica DM2500).
11. An inverted microscope (Nikon Eclipse Ti) is used to record
fluorescent expressions of cells cultured off-chip.
12. Two-sided student T-test is used to determine the significance
for data with Gaussian distribution and equal variances. Groups
with p-values <0.05 are considered significant difference.
4 Notes
1. As the NK cells are suspension cells, dielectrophoresis (DEP)
force is applied to trap cells on the nanochannel before run
nanoelectroporation. Details of DEP for cell capture are
provided in [13]. To maximize ▽E
2 on the NEP device and
to minimize the slew rate of the circuit, the operational amplifiers in the circuit are configured for open loop operation, and a
square wave feed is selected over other functions.
2. For a successful cell electroporation, the condition of threshold
voltage greater than resting transmembrane potential should
be met.
3D Nanochannel Electroporation for Macromolecular Nucleotide Delivery
75