2.9 Quantitative Real-Time PCR
Total RNA was extracted from tumor cells or tissues using TRIzol reagent
(Takara, Tokyo, Japan) according to the manufacturer’s protocol.
The concentration and purity of total RNA were determined using a
Nanodrop ND-1000 spectrophotomer (Thermo Fisher Scientific, Wilmington,
DE, USA), with an A260/A280 ratio between 1.8 and 2.0. Complementary DNA
(cDNA) was synthesized using cDNA Reverse Transcription Kit
(Takara, Tokyo, Japan). Quantitative Real-Time PCR (qRT-PCR) was
performed using SYBR Premix Ex Taq (Takara, Tokyo, Japan) according to
the manufacturer’s instructions in a QuantStudio®3 Real-Time PCR
System (Applied Biosystems, Foster City, CA, USA). The expression levels
of STAT3 downstream target genes were calculated using the
2−ΔΔCT method.
GAPDH was used as an internal reference for the relative
quantifications. For the total RNA extraction of tissues, a small
portion of tumor tissues was removed, frozen in liquid nitrogen,
and homogenized in the reagent described above with 5 mm stainless steel
beads (QIAGEN Inc., Valencia, CA, USA) in the TissueLyser system (QIAGEN
Inc.). The remaining procedures were then performed as described above.
The primers used were as follows: VEGF forward,
5’-CGAGATAGAGTACATCTTCAAGC-3’, and reverse,
5’-TTGATCCGCATGATCTGCATGG-3’; IL6 forward,
5’-AAATGCCAGCCTGCTGACGAAC-3’, and reverse,
5’-AACAACAATCTGAGGTGCCCATGCTA-3’; CASP3 forward,
5’-AACTGGACTGTGGCATTGAG-3’, and reverse,
5’-ACAAAGCGACTGGATGAACC-3’; BCL2 forward,
5’-AGACCGAAGTCCGCAGAACC-3’, and reverse, 5’-GAGACCACACTGCCCTGTTG-3’;ICAM1 forward, 5’-TCAAAAGTCATCCTGCCCCG-3’, and reverse,
5’-TGCTCAGTTCATACACCTTCCG-3’; CCND1 forward,
5’-TCTACACCGACAACTCCATCC-3’, and reverse, 5’-TTCCACTTGAGCTTGTTCACC-3’;MMP9 forward, 5’-AGACGGGTATCCCTTCGACG-3’, and reverse,
5’-AAACCGAGTTGGAACCACGAC-3’; MMP2 forward,
5’-GCCCCAGACAGGTGATCTTG-3’, and reverse, 5’-GCTTGCGAGGGAAGAAGTTGT-3’;MYC forward, 5’-GGCTCCTGGCAAAAGGTCA-3’, and reverse,
5’-CTGCGTAGTTGTGCTGATGT-3’; TXNIP forward,
5’-TCGTGTCAAAGCCGTTAGGA-3’, and reverse, 5’-ATTCTCACCTGTTGGCTGGT-3’;NOP56 forward, 5’-ACAGCTATTCCCGTGCCAAA-3’, and reverse,
5’-CCACTCCCTGACACGCATAG-3’; MFSD2A forward,
5’-AGCTTTGCTATGCACTTGGGG-3’, and reverse, 5’-TCATCCTTCTGAGCCACATCCA-3’;SAT1 forward, 5’-CTCCGGAAGGACACAGCATT-3’, and reverse,
5’-TGATCCTATGCCAAAGCCTCT-3’; F3 forward,
5’-CAGGAAAGAAAACAGCCAAAACA-3’, and reverse, 5’-TACCGGGCTGTCTGTACTCT-3’;CLDN1 forward, 5’-ATTTCTTCTTGCAGGTCTGGCT-3’, and reverse,
5’-GGGGACAGGAACAGCAAAGT-3’; SLC12A3 forward,
5’-CGCACCTTTGGCTACAACAC-3’, and reverse, 5’-GTGTCTGCCTTCCTGCTTGA-3’;HSPA8 forward, 5’-GCCTACACCCCAGCAACCAT-3’, and reverse,
5’-TTGGAGTGGTTCGGTTTCCC-3’; TRIM31 forward,
5’-CGGAAGCGGGGAAACACTAT-3’, and reverse, 5’-CTGAAACTCTTCACTTCTGCAC-3’;MN1 forward, 5’-GGTACATGCCCGCTGACAA-3’, and reverse,
5’-GAGGTCGTGGGCTTCTTTGCT-3’; GAPDH forward,
5’-CGACAGTCAGCCGCATCTT-3’, and reverse, 5’-CCGTTGACTCCGACCTTCA-3’.