RNA isolation and real-time PCR
Total RNA was isolated and purified by using a QIA shredder (QIAGEN,
Tokyo, Japan) and RNeasy Mini kit (QIAGEN). Complementary DNA (cDNA) was
synthesized using the following protocol. One microgram (μg) of total
RNA, a random primer (Takara Bio, Shiga, Japan), dNTP mix (Invitrogen),
and distilled water were mixed and heated to 65℃ for 5 minutes using the
Gene Atlas 485 (ASTEC, Fukuoka, Japan). SuperScript™ Ⅲ RT (Invitrogen),
5× First-Strand Buffer (Invitrogen), 0.1 M DTT (dithiothreitol,
Invitrogen), and RNaseOUT™ (Invitrogen) were then added to the mixture,
which was allowed to incubate at 55℃ for 60 minutes and again at 75℃ for
15 minutes using Gene Atlas 485. cDNA, QuantiTect SYBR Green PCR
(QIAGEN), a specific primer, and distilled water were mixed and
incubated as follows: 45 cycles at 95℃ for 15 seconds, 55°C for 15
seconds, and 75℃ for 20 seconds. The ⊿⊿CT (delta-delta CT) relative
value method, using GAPDH as the housekeeping gene, was utilized to
calculate gene expression, after which the threshold cycle numbers were
obtained using Stratagene Mx3000p (Agilent Technologies, Tokyo, Japan).
The sequences of the specific primers were: CCL5 forward: 5’-TGA CCA GGA
AGG AAG TCA GC-3’, reverse: 5’-AGC CGA TTT TTC ATG TTT GC-3’, GAPDH
forward: 5’-TGA ACG GGA AGC TCA CTG G-3’, reverse: 5’-TCC ACC ACC CTG
TTG CTG TA-3’, TLR3 forward: 5’-CTC AGA AGA TTA CCA GCC GCC-3’, reverse:
5’-CCA TTA TGA GAC AGA TCT AAT G-3’, ICAM-1 forward: 5’-GGC TGG AGC TGT
TTG AGA AC-3’, reverse: 5’-ACT GTG GGG TTC AAC CTC TG-3’.