HUVECs were cultured at a density of 5,000 cells/cm2 at 37 °C and starved overnight. Subsequently, they were incubated with ANG protein (final concentration 1 μg/ml ) or PBS for 6 hr.
Growth protocol
Human umbilical vein endothelial cells (HUVECs), passages 3 to 8. HUVECs were cultured in modified endothelial cell growth medium containing 52% M199 medium (Life Technologies), 36% Human Endothelial-SFM (Life Technologies), 10% fetal bovine serum (Life Technologies) and 15 μg/ml endothelial cell growth supplement (Millipore).
Extracted molecule
total RNA
Extraction protocol
Collect cells with trypsin and washed with PBS, then resuspend in PBS at a density of 2×10E7/ml. Take 10 ul cell suspension and lyse in 90 ul lysis buffer (Tris 15 mM, NaCl 10 mM, MgCl2 3 mM, NP-40 0.3%) for 10 min on ice, and then heat at 99°C for 10 min. Sit the lysate on ice after heating. Take 6 ul lysate as the reverse transcription template. Reverse transcription was performed using Human MegaPlex RT Primer pools. cDNA was amplified using human MegaPlex PreAmp Primer Pools and PreAmp Master mix.The amplified samples were loaded into Taqman microRNA arrays Pool A (377 miRNAs) and pool B (373 miRNAs). Analysed using the 7900 Real Time PCR System (Applied Biosystems)
Label
FAM
Label protocol
n/a
Hybridization protocol
n/a
Scan protocol
n/a
Description
Control
Data processing
Ct values were calculated by SDS 1.2 software (Applied Biosystems) and normalized to MammU6 (miRNA) housekeeping Ct values and expressed as 2-(Ct[microRNA]—Ct[U6]) Fold Change worksheet reports test/control (i.e., ANG/Control) ratios.
