Sybr green premix dimereraser

RNAiso Plus (Takara, Otsu, Japan) was used to isolate mRNA from cells or tissue.
The PrimeScript RT Master Mix (Takara, Otsu, Japan) was used to generate cDNA.
qPCR was performed using a SYBR Green Premix DimerEraser (Takara, Otsu, Japan)
on the 7900HT Fast Real-Time PCR system (Applied Biosystems). All reactions had
a melting curve with a single peak. The cycle threshold (CT) values for the
triplicate samples were averaged and the data were analyzed using the
ΔΔCT method, where fold
change = 2^−ΔΔCT.
All data analyzed were normalized to beta-Actin or GAPDH expression. For the
specific primer sequences (Sangon Biotech, Shanghai, China), see Supplementary Table S4.

Pancreatic cancer cells SW1990, BxPC-3, CaPan-2 and SU.8686 were cultured in media with different concentrations of glutamine (0 and 2 mM). RT-qPCR was performed to evaluate the relative expression levels of
IL6R and
CXCR4. Total RNA was isolated from cell lines using the EZ-press RNA Purification kit (EZBioscience, Shanghai, China), followed by cDNA synthesis using a Color Reverse Transcription kit (EZBioscience). The reverse transcription of RNA into cDNA was performed according to the manufacturer’s protocol. The product were amplified using SYBR-Green Premix DimerEraser (Takara, Dalian, China) on theLightCycler480 II system (Roche, Basel, Switzerland). The relative expression was determined using the 2
^‒ΔΔCq method. qPCR was performed under the following conditions: initial denaturation at 95°C for 3 min followed by 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, with a final extension at 72°C for 1 min.
β-Actin was selected as an internal reference, to compare gene expression in different samples. The following primers were used:
IL6R (forward: 5′-GGCTCTGAAGGAAGGCAAGA-3′, reverse: 5′-CTGGCATCTGGTCGGTTGT-3′);
CXCR4 (forward: 5′-CAGTGAGGCAGATGACAG-3′, reverse: 5′-ACAATACCAGGCAGGATAAG-3′); and
β-actin (forward: 5′-CGTGCGTGACATTAAGGAAGAGT-3′, reverse: 5′-GGAAGGAAGGCTGGAAGAGT-3′).