INTRODUCCIÓN: REVISIÓN CRITICA DEL PROBLEMA

CAPÍTULO IX
primers and MMLV reverse transcriptase (Thermo Fischer) incubated for 50 min at
37 ºC and 15 min at 42°C followed by 5 min at 95°C to inactivate the transcriptase.
The single stranded cDNA (1-2 μL) were amplified by quantitative PCR with a real-
time detection thermal cycler (Chromo4 PTC-200, MJ Research Bio-Rad) in a
reaction mixture (20 μL) containing 10 μL FastStart SYBR Green Master (Roche),
and 100 nmol · L -1 of each primer. SYBR Green I fluorescence was measured after
each amplification cycle. Melting curves were made at the end of each PCR
experiment from 70 to 95 ºC with steps of 1 ºC and 1 s. The mRNA levels for each
gene were determined by quantitative real-time PCR performed in duplicate. The
relative expression ratio was calculated by the comparative threshold method
using β-actin as housekeeping gene from the following equation: R = 2-R = 2 -[ΔCt
sample – ΔCt control] , where Ct is the cycle threshold value. Gene database accession
numbers, sequences for sense (S) and antisense (A) primers, PCR conditions,
cycle counts, and amplification fragment lengths are indicated in Table 1.
c) Nucleus-cytoplasm protein extraction
After the treatments of 300,000 HUVEC per well (6 wells plate), cell culture
medium was aspirated and cells washed with PBS. Cells were detached with a
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