Sybr green real time pcr master mix kit

Total RNA was extracted from individual groups of cells using trizol and reversely transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Takara Biotechnology), according to the manufacturer's instruction. The relative levels of DLG5 to control GAPDH mRNA transcripts were determined qRT‐PCR using the SYBR^® Green Real‐Time PCR Master Mix kit (Thermofisher) and specific primers (Takara) in the QuantStudio 6 Flex Real‐time PCR system (Applied Biosystems). The sequences of primers were GAPDH forward 5′‐CTC CTC CAC CTT TGA CGC TG‐3′ and reverse 5′‐TCC TCT TGT GCT CTT GCT GG‐3′; DLG5 forward 5′‐CTG CAC ATC AAC CTC AGT GG‐3′ and reverse 5′‐CGG CAG CAT ACA CTC CATT‐3′. The results were analysed by 2^−ΔΔCt.

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, United States). cDNA was synthesized using a Reverse Transcription Kit (Promega, Madison, WI, United States). cDNA was used for quantitative PCR reactions to determine the expression of specific genes with SYBR Green Real-Time PCR Master Mix Kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. GAPDH mRNA was applied as endogenous control. Primer sequences are shown in Supplementary Table 1. The total cycles for RT-PCR was set as 40.

Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA using a Reverse Transcription Kit (Promega, Madison, WI). Quantitative PCR reactions were performed using SYBR Green Real-Time PCR Master Mix Kit (Invitrogen). The experiment was performed in triplicate and the relative expression was calculated with the 2^−ΔΔCT method. GAPDH was applied as endogenous controls. Primer sequences are shown in Supplementary Table 2.

RNA was extracted from cells using TRIzol (15596-026, Ambion, Rockford, IL) and cDNA generated using a Reverse Transcription Kit (#600559, Agilent Technologies), according to the manufacturer’s protocol. Quantitative analysis was performed using SYBR Green Real-Time PCR Master Mix Kit (#4472908, Invitrogen, Carlsbad, CA). The experiments were performed in triplicate for each sample and normalized to the housekeeping gene expression PUMILIO. The real time thermocycler Stratagene, model Mx3000P was used, using the program MxPro v2.0. The analysis was performed using the ΔΔCt method^{59 (link)}. PCR was performed using the following primer sets: ZEB1, (forward) 5′-TTCACAGTGGAGAGAAGCCA-3′, (reverse) 5′-GCCTGGTGATGCTGAAAGAG-3′; ZEB2, (forward) 5′-ATAAGGGAGGGTGGAGTGGA-3′, (reverse) 5′-CGCGTTCCTCCAGTTTTCTT-3′; SNAIL (forward) 5′-TTCCAGCAGCCCTACGACCAG-3′, (reverse) 5′-GCCTTTCCCACTGTCCTCATC-3′; SLUG, (forward) 5′-CATGCCTGTCATACCACAACC-3′, (reverse) 5′-CTTGGATGAGGTGTCGGATG-3′; CDH1, (forward) 5′-GAACGCATTGCCACATACAC-3′, (reverse) 5′-ATTCGGGCTTGTTGTCATTC-3′; SDC-1, (forward) 5′-GCCGCAAATTGTGGCTACT-3′, (reverse) 5′-GGTTCTGGAGACGTGGGAATAG-3′; and PUMILIO, (forward) 5′-CGGTCGTCCTGAGGATAAAA-3′, (reverse) 5′-CGTACGTGAGGCGTGAGTAA-3′.