Syber green master mix

The primers of quantitative real time PCR Syber Green method were designed for complete cDNA of Bcl-2, Bax, and β-actin based on sequence data available on NCBI databases utilizing Beacon Designer (PREMIER Biosoft International, Palo Alto, CA; version 7). Primers’ specificity were checked by BLAST analysis (NCBI). A quantitative real-time PCR was done by Syber Green Master Mix (Takara Biotechnology, Otsu and Shiga, Japan). The primers’ specificity and reliability was approved via performing endpoint PCR and amplicon sequencing through DNA sequencing, Applied Biosystems (SEQLAB, Germany). The designed primers are summarized in Table 1. β-actin was utilized as housekeeping gene for the normalization of data. Relative two standard curves real time PCR Syber Green method analyzed gene expressions in a Rotor Gene Q 6000 (Qiagen Hilden, Germany). The data were analyzed using the 6-point 10-fold serial dilution standard curves for genes of interest and reference gene. Standard curves were plotted for target and reference genes, and then Rotor Gene 6000 software (Qiagen, Germany) was used to analyze the data. The relative quantity of Bcl-2 and Bax gene expressions were normalized to the β-actin mRNA relative quantity and expressed as gene expression index.

Total RNA was extracted from the whole blood using TRIzol reagent (category number 15596026; Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed to measure the expression of NKAα₂, NKAα₃, α-spectrin and drebrin using SYBER Green mastermix (Takara, Shiga, Japan) with CFX96 real-time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The sequence for NKAa₂: 5′-CTTTGCATTGACCTGGGCAC-3′, antisense 5′-TTGTCCGTCTGGGAGTTTCG-3′; NKAa3: 5′-CAGGAGAGCCACGGACAAGAA-3′, anti-sense 5′-TTGTCCGTCTGGGAGTTTCG-3′; aspectrin: 5′-GGCTGATGGGATTCTAGGAC-3′, antisense 5′-AACCTGGCAAGATAAAATGTGTC-3′; drebrin: 5′-ACTCAAAAGGAGGGGACCCA-3′, antisense 5′-TACAGGAGGCGGAACCTTTG-3′. GAPDH gene was used as an internal control using the sense primer 5′-GCACCGTCAAGGCTGAGAAC-3′ and antisense primer 5′-TGGTGAAGACGCCAGTGGA3′ (primers were obtained from Sigma-Aldrich Co.). The comparative threshold (CT) cycle values were derived from qbasePLUSsoftware. The ΔCT method of relative quantification was used to determine the fold changes in expression (ΔCT = ΔCT reference – ΔCT target).