Quantitative real-time RT-PCR and western blotting analysis
Extraction of total RNA of tissues and cells, quantitative real-time PCR
and western blotting were performed as described previously
(He et al., 2014). For quantitative
real-time RT-PCR, total RNA of placental tissues and cells were
extracted by TRIzol reagent (Invitrogen). 2 µg RNA was reverse
transcribed to generate cDNA by superscript reverse transcriptase
(Invitrogen). Quantitative real-time PCR was carried out using
MiniOpticon™ Real-Time PCR Detection System (BioRad, Hercules, CA).
Real-time PCR reaction solution consisted of 2.0 μl diluted cDNA, 0.2 μM
of each paired primer and 1×PCR Master Mix (TaKaRa, Otsu, Japan). SYBR
Green (Roche Ltd, Basel, Switzerland) was used as detection dye.
Amplification of the housekeeping genes β-actin was measured for each
sample as an internal control for sample loading and normalization. The
primers used were listed as followings: sFlt1 (NM_001159920), sense:
TTGGGACTGTGGGAAGAAAC; anti-sense: TTGGAGATCCGAGAGAAAACA. β-actin
(NM_001101), sense: TGTGTTGGCGTACAGGTCTTTG; anti-sense:
GGGAAATCGTGCGTGACATTAAG. The temperature range to detect the melting
temperature of the PCR product was set from 60–95℃. The specificity of
PCR products was examined by melting curve at the end of the
amplification and subsequent sequencing. To determine the relative
quantitation of gene expression for both sFlt1 and β-actin genes, the
comparative Ct (threshold cycle) method with arithmetic formulae
(2-ΔΔCt) was used.
For the western blotting analysis,
70 mg of tissues were homogenized in
cold RIPA lysis buffer containing protease inhibitor cocktail tablet
(Roche, Indianapolis, IN) and primary cultured cells were scraped off
the plate in the presence of RIPA lysis buffer containing protease
inhibitor cocktail tablet. The protein concentration was quantified by
BCA kit. Then lysates were quickly sonified in ice bath, boiled 5 min at
100ºC, and stored at -80ºC. 30 µg of protein samples were separated by
10% SDS-PAGE and subsequently transferred to nitrocellulose membranes.
After blockage in 5% skim milk powder in 0.1% Tris-buffered
saline/Tween 20 (TBST), membranes were incubated with primary antibodies
for 11β-HSD1 (Santa Cruz, Cat# sc-19259, RRID: AB_2119513, Clone ID:
C-17), 11β-HSD2 (1:2000; Abcam,
Cat# ab80317, RRID: AB_1658782C), a disintegrin and metalloproteinase
(ADAM)17 (1:2000; Abcam, Cat#
ab2051 RRID: AB_302796) , ADAM10
(1:2000; Abcam, Cat# ab124695
RRID:AB_10972023, Clone ID: EPR5622) and GAPDH(1:2000; Abcam, Cat#
ab181602, RRID:AB_2630358, Clone ID: EPR16891) at 4 °C overnight. Then,
the membrane was incubated with a secondary horseradish
peroxidase-conjugated antibody (Proteintech Inc,WuHan,China) for 1 h
at room temperature. Immunoreactive proteins were visualized using the
enhanced chemiluminescence Western blotting detection system (Santa
Cruz). The chemiluminiscent signal from the membranes was quantified by
image J software. To control sampling errors, the ratio of band
intensities to GAPDH was obtained to quantify the relative protein
expression level.