Sybr green pcr master mix kit

Total RNA was extracted from RAW264.7 cells and lung tissue samples using TRIzol reagent (Thermo Fisher Scientific). The first strand cDNA was synthesized by reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR was performed using the SYBR Green PCR Master Mix Kit (Thermo Fisher Scientific). The PCR primer sequences are summarized as follows: TNF-α forward: 5′-CCACCACGCTCTTCTGTCTAC-3′, reverse: 5′-AGGGTCTGGGCCATAGAACT-3′; IL-6 forward: 5′-AGTTGCCTTCTTGGGACTGA-3′, reverse: 5′-CTGTGAAGTCTCCTCTCCGG-3′; MCP-1 forward: 5′-CTTCTGGGCCTGCTGTTCA-3′, reverse: 5′-CCAGCCTACTCATTGGGATCA-3′; SIRT1 forward: 5′-AGTTCCAGCCGTCTCTGTGT-3′, reverse: 5′-CTCCACGAACAGCTTCACAA-3′; GAPDH forward: 5′-TCACCACCATGGAGAAGGC-3′, reverse: 5′-CCTAAGCAGTTGGTGGTGCA-3′. The relative gene expression was calculated by normalizing against GAPDH.^{17 (link)}

Total RNA was isolated from cells or tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Cergy Pontoise, France). Next, 3 mg total RNA was denatured for 10 min at 70°C and then reversed transcribed into cDNA at 37°C for 90 min using 300 U Moloney murine leukemia virus reverse transcriptase, 15 mg oligo dT primers (both Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1 mM deoxynucleoside triphosphate (Bioline, London, UK) in a total volume of 30 ml. qPCR was then performed using a SYBR Green PCR Master Mix kit (ABgene; Thermo Fisher Scientific, Inc., Courtaboeuf Cedex, France) supplemented with 0.5 mM primers. The PCR mixture contained 7.5 µl SYBR Green, 4.5 µl water, 1 µl forward and reverse primers, respectively, and 2 µl DNA template. The primers were: Human TMEM35 forward, 5′-TGGGGACTATCAAGCTGACC-3′, and reverse, 5′-CAATGCTTTTTCGGAGGAGA-3′; β-actin forward, 5′-AATCGTGCGTGACATTAAGGAG-3′, and reverse, 5′-ACTGTGTTGGCGTACAGGTCTT-3′. The thermal cycling conditions used were as follows: 95°C for 15 min, then 40 cycles at 95°C for 20 sec, 58°C for 15 sec, and 72°C for 15 sec. Signals with a threshold cycle (Cq) value of >39 were considered to indicate no transcription of the target gene. The relative expression of mRNA was calculated using the 2^−ΔΔCq method (25 (link)).

The total RNA in the transfected CRC cells was isolated using the one-step TRIzol method, then the RNA was purified. Total RNA was then analyzed for concentration and purity using NanoDrop (Thermo Fisher, Waltham, MA, USA). Complementary DNA (cDNA) was prepared using a reverse transcription (RT) kit. We took 2 µL of the RT product as template, GAPDH as the internal reference, and used SYBR Green PCRMaster Mix kit (Thermo Fisher) for quantitative polymerase chain reaction (qPCR) detection. Each reaction system in the 20 µL vial included 2 µL of RT product, 10 µL of 2× SYBR Green dye, 0.8 µL of both primers (forward and reverse), and 6.4 µL of double distilled water (ddH2O). The 45 cycles of the reaction consisted of 95 ℃ for 5 minutes, 60 ℃ for 30 seconds, and at 72 ℃. for 10 seconds. After the reaction, the relative quantitative 2^-ΔΔCt method was used for analysis.