Real time PCR
PCR amplification of TMEM187,
CLACA1,
SERPINB2,
POSTN and FKBP51 genes and the
house keeping gene GAPDH were carried out in separate PCR tubes using
gene specific primersas follows: TMEM187: forward
5′-ACTTATGCCGCCAGTATCTCG-3′′; reverse 5′-AGGCGCAAAAGATGGACCAG-3′12 ;
CLACA1: forward 5′-ATGGCTATGAAGGCATTGTCG -3′; reverse
5′-TGGCACATTGGGGTCGATTG -3′ 13 ; SERPINB2:
forward 5′-GCTGGAGATGTTAGCATGTTCTTG-3′; reverse
5′-GGCTTGGTGGAACACTTCAGAAAG-3′ 14 ;
POSTN: forward
5′-GACTCAAGATGATTCCCTTT -3′; reverse 5′-GGTGCAAAGTAAGTGAAGGA-3′15 ;
FKBP51: forward
5′-GGATATACGCCAACATGTTCAA-3′; reverse 5′-CCATTGCTTTATTGGCCTCT-3′16 ; and
GAPDH: forward
5’-TGCACCACCAACTGCTTAGC-3’; reverse 5’-GGCATGGACTGTGGTCATGAG-3’17 QuantiTect® SYBR® Green PCR kit supplied
by Qiagen; Germany was used for amplification. The amplification
reaction contained 10 μL of 2x QuantiTect SYBR Green PCR Master Mix, 2
μL of cDNA, 0.5 μL of the forward primer, 0.5 μL of the reverse primer
and Nuclease-free H2O up to 20 μL final volume. The thermal profile of
the PCR reaction was 95°C for 15
min., 40 cycles of 94°C for 15 sec, annealing temperature of the gene
(55°C for TMEM187, 54°C for
CLACA1, 54°C for SERPINB2, 47°C for POSTN, 51°C for FKBP51 and 57°C for
GAPDH) for 30 sec and 72°C for 30 sec.
Melting curve analysis using StepOne software (Applied Biosystems, USA)
was performed to ensure specificity of the amplification products.
Relative expression of the target genes in each sample were finally
detected after normalization to GAPDH expression and calculated as
2^-ΔCt.18