Taqman universal pcr master mix kit

Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Next, 1 μg of total RNA was subjected to reverse transcription PCR using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) according to manufacturer’s protocol. The thermocycling conditions were: 60 min at 42 °C, followed by 15 min at 70 °C, and 30 min at 4 °C. qRT-PCR was performed using a TaqMan Universal PCR Master Mix kit (Applied Biosystems) in an Bio-Rad iQ5 Real-Time PCR System and U6 or β-action was used as an endogenous control for miR-222-3p and CD4 respectively. Reactions were performed in triplicate. The thermocycling conditions for amplifying miR-222-3p were: 95 °C for 30 s, followed by 45 cycles of 5 s at 95 °C and 60 °C for 30 s, and concluded with 1 min at 60 °C. Meanwhile, the thermocycling conditions for amplifying CD4 were: 95 °C for 30 s, followed by 45 cycles of 5 s at 95 °C and 57 °C for 20 s, and concluded with 1 min at 60 °C [10 (link)]. After finalization of the qRT-PCR experiments, the average values of the cycle threshold (Ct) of the reactions in triplicate were determined. Data analysis was performedusing the 2^-ΔΔCt method.

The expression levels of miR-9 in osteosarcoma and corresponding non-cancerous tissues were detected using a qRT-PCR assay. Briefly, total RNA from tissue samples was extracted with TRizol reagent (Invitrogen, Breda, the Netherlands) according to the manufacturer’s instructions. cDNA was reverse transcribed from total RNA samples using specific miRNA primers from the TaqMan MicroRNA Assays and reagents from the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Products were amplified by PCR using TaqMan Universal PCR Master Mix kit (Applied Biosystems). The quantitative PCR was performed with the specific primers as follows: miR-9_F, 5′-GTGCAGGGTCCGAGGT; miR-9_R, 5′-GCGCTCTTTGGTTATCTAGC-3′; U6_F, 5′-CTCGCTTCGGCAGCACA-3′; U6_R, 5′-AACGCTTCACGAATTTGCGT-3′. Small nucleolar RNA U6 was used as an internal standard for normalization. The cycle threshold (C_T) was calculated. The 2^-ΔCT (ΔC_T = C_TmiR-9 – C_{TU6 RNA}) method was used to quantify the relative amount of miR-9. In addition, each measurement was performed in triplicate.

The total RNA was extracted from ~ 1 cm² of adipose tissue using QIAzol lysis reagent (Qiagen, Hilden, Germany). The amount of RNA was determined using NanoDrope (Thermo Scientific, Waltham, MA, US) spectroscopy, and diluted to a final concentration of 50 ηg/μl in 20 μl. In total, 1 μl of RNA was used for the synthesis and amplification of complementary DNA (cDNA), which followed the protocol of the high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, US). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using the PPAR gamma gene (Mm00440940_m1), and the endogenous control GAPDH (Mm99999915_g1) followed the TaqMan Universal PCR Master Mix Kit (Applied Biosystems) using the StepOne Real-Time PCR System (Life Technologies, Foster City, CA, US). Real-time PCR reactions were conducted as follows: after a pre-denaturation and polymerase-activation program (2 minutes at 50°C and 10 minutes at 95°C), 50 cycles, each one consisting of 95°C for 15 seconds and of 60°C for 1 minute. The negative controls consisted of wells in which the cDNA was absent. The relative expression of PPAR gamma/GAPDH was calculated using the equation ΔCt, which expresses the difference between the number of threshold cycles (Cts) of the target genes and the endogenous control.