Veriti 96 well pcrthermal cycler

The salting out method was used to extract DNA from
peripheral blood samples. Genotyping was performed using
Allele specific polymerase chain reaction (AS-PCR) methods
(10 ). We used primers from the previous study (11 (link)) and the
sequence of four primers (synthesis by metabion international
AG, Germany) was shown as follows:
Arginine-based (G) allele:
F: 5´-TCCCCCTTGCCGTCCCAA-3´
R: 5´-CTGGTGCAGGGGCCACGC-3´
Proline-based (C) allele:
F: 5´-GCCAGAGGCTGCTCCCCC-3´
R: 5´-CGTGCAAGTCACAGACTT-3´
PCR was done in a final volume of 10 μl reaction for each
allele containing: Taq DNA Polymerase 2x Master Mix
RED (Ampliqon, Denmark), 1 μl genomic DNA (200-300
ng), 1 μl of each primer (10 μM, Metabion International
AG, Germany) and adequate DNase free water (Sinaclon,
Iran). Amplification temperature stages were performed
for 5 minutes at 95˚C and then 35 cycles including 30
seconds at 94˚C, 30 seconds at 63˚C, 30 seconds at 72˚C,
followed by 7 minutes at 72˚C in a Veriti 96 well PCR
Thermal Cycler (Thermo Fisher Scientific).

Tissues were homogenized in RNALater and purified using MagMAX-96 for Microarrays Total RNA Isolation Kit (Ambion by Life Technologies). Genomic DNA was removed using RNase-Free DNase Set (Qiagen). mRNA was reverse-transcribed into cDNA using SuperScript VILO Master Mix (Invitrogen Life Technologies) and Veriti 96-well PCR Thermal Cycler (Thermo Fisher Scientific). cDNA was amplified with SensiFAST Probe Hi-ROX (Meridian Life Science) using 12k Flex System (Applied Biosystems). PCR reactions were done in triplicate. GAPDH was used to normalize cDNA input differences. Data reported as comparative CT method using delta delta CT. Probes listed in the Supplementary Materials.