Realq pcr master mix

The quantitative real‐time PCR (qRT‐PCR) reaction was performed in 96‐well plates using 2.0X RealQ‐PCR Master Mix^® with SYBR Green (Ampliqon) on ABI StepOnePlus™ Real‐Time PCR Detection System (Applied Biosystems). All qRT‐PCR primers were designed by Allele ID 6 software (Premier Biosoft) and described in Table 1. Each reaction mixture consisted of 1 µL cDNA (10 ng), 10 µL 2X RealQ‐PCR Master Mix^®, 1 µL (10 pmol/µL) of both forward and reverse primers, and 7 µL of PCR‐grade water, equating to a final volume of 20 µL. The beta‐2 microglobulin (β2M) mRNA was used as the reference gene. β2M was selected as the reference gene according to previous research for identification of housekeeping control genes in colorectal cancer.¹⁹ The thermal profile of the reaction was performed using the following conditions: initial denaturation at 95°C for 15 minutes; followed by 40 cycles at 95°C for 15 seconds and 60°C for 60 seconds followed by melting curve stage assessment. The melting curve profile and agarose gel electrophoresis were performed to verify the specificity of primers and the authenticity of the PCR products.

Synthesis of cDNA and isolation of total RNA was carried out using the kits (SinaClon, Tehran, Iran), based on instructions prepared by the manufacturer. The qPCR reactions were conducted utilizing RealQ PCR Master Mix (A320799 Ampliqon, Denmark) in Rotor-Gene 6000 Real-Time Thermal Cycler (Corbett Research, Australia). The primers for HPRT, NANOG, collagen I, and collagen III were purchased from the Stem Cell Technology Research Center, Iran. LinReg PCR application (Amsterdam, Netherlands) was utilized for primer quality evaluation. Relative Expression Software Tool (REST 2009, Corbett Research, Australia) was used to measure relative gene expression through the 2^-∆∆Ct approach.

The optic chiasmata were collected from the mice brains for total RNA isolation using the
RiboEx solution (Gene All, Korea) as stated in the manufacturer’s protocol. Reverse
transcription and cDNA production were performed by a cDNA reverse transcription Kit
(Parstous Biotechnology, Iran) based on the manufacturer’s instructions. The produced cDNA
was used for analysis of gene expression. Real-time polymerase chain reaction (q-PCR) was
performed by a Real q-PCR Master Mix (Ampliqon, Denmark) on a Rotor-Gene device (Qiagen,
Germany). All reactions were performed in duplicate. The relative amount of mRNA was
calculated using the delta-delta cycle of threshold (Ct) method, and normalization was
done using Gapdh as a housekeeping gene. Primer sequences are shown in
Table 1.