Predictive genetic markers of coagulation, inflammation and apoptosis in Perthes disease Serbian experience
European Journal of Pediatrics
Sanja Srzentic, Gordana Nikcevic, Dusko Spasovski, Zoran Bascarevic, Zorica Zivkovic,
Zorica Terzic-Supic, Dragana Matanovic, Valentina Djordjevic, Sonja Pavlovic and Vesna
Spasovski*
*Corresponding author
Vesna Spasovski
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade
Vojvode Stepe 444a, 11010 Belgrade, Serbia
e mail: vesna.spasovski@imgge.bg.ac.rs
Supplementary material
TNF-α G-308A gene variant detection
Gene variant TNF-α -308G>A (rs1800629) was detected by PCR-RFLP technique.
PCR fragment was synthesized using following set of primers: (forward: 5'AGGCAATAGGTTTTGAGGGCCAT-3', reverse: 5'-ACACTCCCCATCCTCCCTGCT-3').
The forward primer sequence was altered (underlined base) to generate a restriction site in the
major allele. After digestion of PCR product with NcoI (MBI Fermentas, Vilnius, Lithuania)
fragments of the following length were obtained: for genotype GG- 97+20bp, GA117+97+20bp and AA- 117bp.
IL-3 C-16T and C132T detection
Direct sequencing of 320bp PCR fragment, containing both polymorphic sites 16C>T (rs31480) and C132T (rs40401) in IL-3 gene, was performed in order to detect these
gene variants. PCR fragment was synthesized using following set of primers: forward: 5'GCCAGGGTAGTCCAGGTGAT-3', reverse: 5'-CATTGAGGTTGTTGAAGTCC-3'.
Sequencing was performed using the BigDye Terminator Sequencing kit (Applied
Biosystems, USA), on ABI PRISM 310 Genetic Analyzer.
Supplementary Table 1 Primers, probe and assays used for qRT-PCR analyses
Gene
Bax
Bcl
Bcl2L12
GAPDH
(Syber)
F: 5' - TGG CAG CTG ACA TGT TTT CTG AC - 3'
R: 5' - TCA CCC AAC CAC CCT GGT CTT - 3'
F: 5' - TCG CCC TGT GGA TGA CTG A - 3'
R: 5' - CAG AGA CAG CCA GGA GAA ATC - 3'
F: 5' - CCC TCG GCC TTG CTC TCT - 3'
R: 5' - GGG CCA CCA AAG CAT AGA AG - 3'
F: 5' – GTG AAG GTC GGA GTC AAC G- 3'
R:5' – TGA GGT CAA TGA AGG GGT C - 3'
Fas
TaqMan Gene Expression Assay - Hs00163653_m1
FasL
TaqMan Gene Expression Assay - Hs00181225_m1
GAPDH
F: 5′ GAAGGTGAAGGTCGGAGT 3′
(TaqMan)
R: 5′ GAAGATGGTGATGGGATTTC 3′
FAM-CAAGCTTCCCGTTCTCAGCC-TAMRA
Abbreviations: F- forward; R- reverse