Analytical Chemistry Chemical Cytometry Quantitates Superoxide

dilution and hybridization buffer. The pH of the TNE buffers was
adjusted to 7.4 by addition of 1 M HCl dropwisely. The buffer
solution was then filtered through a 0.22 µm nylon membrane
filter, autoclaved, and photobleached with UV-C lamp overnight
prior to use.
Preparation of Probe and Target MicroRNA Oligonucleotides.
Commercial available LNA-modified oligonucleotide probe
(miRCURYLNAmicroRNADetectionProbe,Productno:38102-00)
(3′-TCAACATCAGTCTGATAAGCTA-5′) specific to hsa-miR-21
was purchased from Exiqon (Denmark). Two HPLC-purified
synthetic DNA oligonucleotides having the complementary sequence
to hsa-miR-21 (3′-TCAACATCAGTCTGATAAGCTA-5′)
and the mature sequence of hsa-miR-214 (3′-ACAGCAGGCACA-
GACAGGCAGU-5′) were custom-designed and obtained from
Invitrogen (Hong Kong) as the DNA probe and the negative
control of the experiments. Two Anti-miR miRNA inhibitors of
miR-21 (AM17000, Product ID: AM10206, 3′-CAACACCAGUC-
GAUGGGCUGU-5′), and anti-miR-21 (AM17000, Product ID:
AM12979, 3′-UAGCUUAUCAGACUGAUGUUGA-5′), were purchased
from Ambion, acting as the RNA probe and target miR-21
strands, respectively. All oligonucleotides were suspended in 1
µL of DEPC-treated water (Ambion) and further diluted into
appropriate concentration with 1× TNE buffer. The melting
temperatures (Tm) of the oligonucleotides were predicted using
the Probe Tm Predictor accessible online (www.exiqon.com)
based on the thermodynamic nearest neighbor model.
Optimization of Hybridization Conditions. Ionic Strength.
For the determination of optimum hybridization ionic strength,
sodium chloride concentrations from 0 to 500 mM in the TNE
buffer were used throughout the oligonucleotides dilution and
hybridization mixture preparation.
Selection of Probe. For the selection of optimal probe with the
best hybridization affinity toward miRNA, probes of DNA, RNA
and LNA were prepared and hybridized with target miR-21 as
described below. In addition, two control experiments including
probes only and negative control miR-214 were also performed.
Hybridization Time. For the determination of optimum hybridization
time, hybrids of same concentration and ionic strength
(250 mM NaCl) were incubated for 15 min, 30 min, 1 and 3 h
respectively.
Hybridization and Labeling of MicroRNA. The hybridization
and fluorescence labeling of miRNAs were performed according
to the following procedures. LNA probe, DNA probe, RNA
probe, miRNA target, and DNA negative control oligonucleotides
were diluted with 1× TNE buffers (pH 7.4) to 300 pM in
concentration, respectively. The hybridization cocktail contained
15 µL of 300 pM probe strand, 15 µL of appropriate concentration
of target miR-21 strand, and 14 µL of TNE buffer. The cocktail
was incubated in form of free-diffusing solutions in dry bath
(AccuBlock Digital Dry Bath D1100, Labnet, NJ) for 1 h. The
hybridization temperature was set to be 20 °C below the T m of
the probe, that is, 52, 47, and 36 °C for LNA, DNA, and RNA
probe, respectively. After incubation, 1 µL of 100 nM YOYO-1
Iodide (YOYO) (Invitrogen) was added subsequently to label
the hybrids. YOYO labels the hybrids in a dye to base pair
(dye/bp) ratio of 1:1. The mixture was kept for 5 min in order
to achieve equilibrium and 10 µL of solution was pipetted to
the precleaned coverslips for TIRF imaging.
External Calibration of MicroRNA. A calibration curve was
established to correlate number of detected single fluorescent
spots and concentration of miRNAs. Synthetic miRNA with final
concentration of 100, 75, 50, 25, 10, 5, and 1 pM target miR-21
strand was hybridized with probe strand of final concentration of
100 pM under optimal conditions as mentioned above.
Cell Culture. Human umbilical vein endothelial cells (HU-
VEC) were purchased from Lonza (Walkersville, MD). M199
medium, heparin, gelatin, and endothelial cell growth supplement
(ECGS) were purchased from Sigma. Fetal bovine serum (FBS)
and penicillin-streptomycin (PS) were purchased from Invitrogen.
Dulbecco’s modified Eagle medium (DMEM) and Roswell Park
Memorial Institute medium (RPMI) were purchased from Gibco
(Grand Island, NY). HUVEC were grown in M199 medium
supplemented with 20% heat-inactivated FBS, 20 µg/mL ECGS,
90 µg/mL heparin, 1% PS in 75 cm 2 culture flasks coated with
0.1% gelatin. HUVEC from passage 2-6 were used in this study.
In addition, human hepatocellular carcinoma (HepG2) was
cultured in DMEM supplemented with 10% FBS and 1% PS,
and invasive breast ductal carcinoma (MCF-7) was cultured in
RPMI supplemented with 10% FBS and 0.5% PS. All cells were
maintained in a humidified incubator at 37 °C with 5% CO2 and
80% relative humidity.
Total RNA Isolation. Total RNA of HUVEC, HepG2, and
MCF-7 were extracted using TRIzol Reagent (Invitrogen) according
to the manufacturer’s protocol. Briefly, the sample was lysed
and homogenized with TRIzol Reagent. Phase separation was
followed by addition of chloroform and centrifugation. RNA in the
aqueous phase was then recovered by precipitation with isopropyl
alcohol. The RNA pellet was washed with 75% ethanol and finally
redissolved in RNase-free water. The RNA quantity was determined
by measuring optical density at 260 nm using the NanoDrop
ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington,
DE), and the RNA quality was assessed by performing
agarose gel electrophoresis.
Quantification of miR-21 in Cells by Quantitative Real-
Time PCR (qRT-PCR). Complementary DNA (cDNA) was
generated from 10 ng of total RNA per 5 µL of gene specific
reverse transcription (RT) reaction by using reagents from
TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems,
Foster City, CA) and mature hsa-miR-21-specific RT primer
(5×) from TaqMan MicroRNA Assays (P/N: 4373090, Applied
Biosystems). Each RT reaction contained 1 mM dNTPs, 50 U
MultiScribe reverse transcriptase, 1× reverse transcription buffer,
and 3.8 U RNase inhibitor as well as 1× specific RT primer. The
reaction mixture was incubated in PTC-100 Programmable Thermal
Controller (MJ Research, Inc., Waltham, MA) for 30 min at
16 °C, 30 min at 40 °C, 5 min at 85 °C, and then held at 4 °C. The
cDNA sample was then amplified by PCR using TaqMan 2×
Universal PCR Master Mix (No AmpErase UNG) (Applied
Biosystems) and TaqMan Assay (20×) from TaqMan MicroRNA
Assays (P/N: 4373090, Applied Biosystems). Each PCR reaction
included 0.67 µL of RT product, 5 µL of TaqMan 2× Universal
PCR Master Mix, No AmpErase UNG, and 0.5 µLof20× TaqMan
Assay (a mix preformulated miRNA-specific forward PCR primer,
specific reverse PCR primer and miRNA-specific TaqMan MGB
probe). The reaction mixture was then run at 95 °C for 10 min,
followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min in
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
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