RNA Extraction and Real Time Quantitative Polymerase Chain Reaction (qRT-PCR)

Parts of the solid tumor (groups 3-6) and the right thigh of the lower limb (groups 1 and 2) from animals were used for the determination of B-cell lymphoma 2 (BCL2) and nuclear factor kappa B (NF-κB) gene expression. RNeasy^® Mini kit (Qiagen, Germany) was used to obtain the total RNA from the tissue samples as specified by the manufacturer’s instructions, reverse transcribed into complementary DNA (cDNA) by Thermo Scientific™ RevertAid™ First Strand cDNA Synthesis Kit (Fermentus, Thermo Fisher Scientific Inc, UK), and then amplified using quantitative real-time polymerase chain reaction (qRT-PCR), in a thermal cycler (ABI PRISM 7500 Fast Sequence Detection System, USA), using Power SYBR^® Green PCR Master Mix (Applied Biosystems, USA). The following thermal cycling conditions were used: 95°C for 10 minutes, subsequently, 40 cycles of 95°C for 15 s, finally 60°C for 1 minute. The sequence of PCR primer pairs and gene bank accession numbers were as follows; B-Cell Lymphoma 2 (BCL2) ({"type":"entrez-nucleotide","attrs":{"text":"NM_009741.5","term_id":"929981608","term_text":"NM_009741.5"}}NM_009741.5): F: 5′-GTG GTG GAG GAA CTC TTC AG- 3′, R: 5′ GTT CCA CAA AGG CAT CCC AG-3′, ⁴¹ Nuclear Factor-Kappa B (NF-κB) ({"type":"entrez-nucleotide","attrs":{"text":"NM_008689.2","term_id":"117606363","term_text":"NM_008689.2"}}NM_008689.2): F: 5′-GAA ATT CCT GAT CCA GAC AAA AAC-3′, R: 5′-ATC ACT TCA ATG GCC TCT GTG TAG-3′ and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ({"type":"entrez-nucleotide","attrs":{"text":"NM_008084","term_id":"576080553","term_text":"NM_008084"}}NM_008084), F: 5′-ATG TGT CCG TCG TGG ATC TGA C-3′, R: 5′-AGA CAA CCT GGT CCT CAG TGT AG-3′. ⁴² The obtained data were examined, handling the ABI Prism sequence detection system software, then computed by PE Biosystems (Foster City, CA) v1•7 Sequence Detection Software. Using the proportional threshold cycle technique, the genes’ relative expression ratios were assessed and standardized to the GAPDH gene. The expression 2^−ΔΔCt was applied to quantify the final relative ratios. ⁴³