Sybr green pcr master mix

MOLT3, PEER, and JURKAT cells were cultured and transduced with shLuciferase, shDLST1, or shDLST2 lentivirus as described above. On day 5 post-infection, the cells were harvested and subjected to total RNA extraction using Trizol reagent (15596026, Invitrogen). cDNA was synthesized with a Reverse Transcription Kit (205311, Qiagen, Hilden, Germany) for qPCR.
SYBR Green PCR master mix (QP004, Genecopoeia, Rockville, MD, USA) and a Step-One PCR instrument (Applied Biosystems, Waltham, MA, USA) were utilized for the qPCR reaction according to the manufacturer’s instructions. The qPCR primer sequences included: β-ACTIN (forward: 5′-GATTCCTATGTGGGCGACGA-3′, reverse 5′- AGGTCTCAAACATGATCTGGGT-3′), IDH1 (forward: 5′- CTCTGTGGCCCAAGGGTATG-3′, reverse 5′-GGATTGGTGGACGTCTCCTG-3′), and IDH2 (forward: 5′-ACAACACCGACGAGTCCATC-3′, reverse 5′-GCCCATCGTAGGCTTTCAGT-3′. All reactions were performed in triplicate.

Total RNA was extracted from 67 CRC tissues and 24 normal colorectal tissues using Trizol reagent (Invitrogen Life Technologies). Two micrograms total RNA were subjected to reverse transcription using cDNA Synthesis Kit (Genecopeia, USA). Real-time PCR was performed with SYBR Green PCR master mix (Genecopeia, USA) and ABI 7500 Fast Dx (Applied Biosystems Co. Ltd., USA). All experiments were carried out in triplicate, and the results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following primers were used: TMEM16A, sense primer, 5′-GATCCCATCCAGCCCAAAGTG-3′; antisense primer, 5′-CGGGTTTTGCTGTC GAAAAAGGA-3′; GAPDH, sense primer, 5′-CGGACCAATACGACCAAATCCG-3′; antisense primer, 5′-AGCCACATCGCTCAGACACC-3′. Dissociation curve analysis of all PCR products showed a single sharp peak and the size of each amplified product was confirmed by ethidium bromide-stained agarose gel electrophoresis. TMEM16A was calculated using 2^−ΔCt method. The ΔCt represents the average Ct for the target gene (TMEM16A) minus the average Ct for the reference gene (GAPDH). Values higher than 0.001 were considered positive for mRNA expression. The mRNA expression was further classified as low expression (values between 0.001 and 0.01), medium expression (values between 0.01 and 0.1) and high expression (values above 0.1).