Total RNA was extracted from treated and nontreated RAW264.7 cells or PMs with an RNA extraction kit (Flyjet Bio) according to the manufacturer’s instructions. Complementary DNA was synthesized using an equivalent amount of total RNA (1 μg) in a 10 μl reverse transcriptase reaction mixture using a cDNA synthesis kit (TaKaRa Biomedical Technology, Japan). Gene expression was analyzed by using SYBR Green (MedchemExpress). Gene expression for each sample was normalized to that of β-actin (Actb), and the differences were determined using the 2 ^ - (ΔΔCt) methods. The primer pairs used in this study were listed below: Il6: forward, 5’-AGT CCT TCC TAC CCC AAT TTC C-3’, reverse, 5’-TAA CGC ACT AGG TTT GCC GA-3; Mcp1: forward, 5’-AGC CAA CTC TCA CTG AAG CC-3’, reverse, 5’-TCT CCA GCC TAC TCA TTG GGA -3’; Tnfa: forward, 5’-GTC CCC AAA GGG ATG AGA AGT-3’, reverse, 5’-TTT GCT ACG ACG TGG GCT AC-3’; Actb: forward, 5’-GGC TGT ATT CCC CTC CAT CG-3’, reverse, 5’-CCA GTT GGT AAC AAT GCC ATG T-3’
Sybr green
Manufactured by MedChemExpress
SYBR Green is a fluorescent dye used in real-time PCR (qPCR) for the detection and quantification of DNA. It binds to double-stranded DNA and emits a fluorescent signal that can be measured to determine the amount of DNA present in a sample.
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Lab products found in correlation
2 protocols using sybr green
1
Quantification of Gene Expression in RAW264.7 Cells
2
Profiling PP2A Expression in Prostate Cancer
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Total RNA was extracted from the DU145, PC-3 and LNCaP cell lines. using TRIzol® (Takara Biotechnology Co., Ltd.). SYBR Green was used as the fluorophore (MedChemExpress). Total RNA was reverse transcribed into cDNA using random primers and a RT Master Mix for qPCR (gDNA digester plus), according to the manufacture's protocol (cat. no. HY-K0511; MedChemExpress). The following thermocycling conditions were used for RT: 25°C for 5 mins, 42°C for 40 mins and 85°C for 2 mins. The following thermocycling conditions were used for qPCR: Initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 40 sec, 50°C for 40 sec and 72°C for 1 min and final extension at 72°C for 5 min. PP2A expression was subsequently determined using qPCR and the following primers: Human PP2A forward, 5′-AGAAGAGGATGAACCCACGC-3′ and reverse, 5′-TGCTAGGCTGGAAATCAGGG-3′; and GAPDH forward, 5′-CCTTCCGTGTCCCCACT-3′ and reverse, 5′-GCCTGCTTCACCACCTTC-3′. GAPDH expression was used as the internal control for normalization. The 2−ΔΔCq method was used for the quantification of the expression levels (23 (link)).
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