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Qs5 384 well pcr system

Manufactured by Thermo Fisher Scientific

The QS5 384-well PCR system is a compact, high-throughput real-time PCR instrument designed for gene expression analysis, genotyping, and other PCR-based applications. It features a 384-well plate format and can perform up to 4 experiments simultaneously. The system provides accurate temperature control and optical detection for reliable real-time PCR results.

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2 protocols using qs5 384 well pcr system

1

Quantitative Analysis of miRNA Expression

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Total RNA was extracted using the Zymo Quick-RNA MiniPrep Kit according to the manufacturer’s protocol. For pri- and pre- miRNAs and mRNAs, RT was performed using 200 ng of RNA with a Qscript cDNA Synthesis Kit (QuantaBio). For mature miRNAs, the RT reaction was performed using 200 ng of RNA with the miScript II RT Kit in a total volume of 20 μl (Qiagen). Subsequent qPCR analysis (see Supplementary Table 2 for primer sequences and Supplementary Figs. 27 for primer validation) using Power SYBR Green Master Mix (Life Technologies) or a TaqMan assay and a Applied Biosystems QS5 384-well PCR system (software v.1.3.0). For mature miR-155 levels, TaqMan assays were performed using the ipu-miR-155 Taqman Assay (Thermo Fisher Scientific, 467534). Data were analysed using the ΔΔCt method as described previously13 (link).
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2

Dose-dependent miRNA-200c regulation

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After transfection, cells were allowed to recover for 8 h in complete growth medium before splitting into 12-well plates (100,000 cells/well). After adhering for 12 h, the cells were treated with 1 or TGP-200c in dose response, vehicle (DMSO), LNA-200c, or LNA-Scramble for 60 h. Total RNA was extracted using the Zymo Quick-RNA Miniprep Kit per the manufacturer’s protocol, including DNase treatment. Reverse transcription (RT) for mature miRNAs was completed with 300 ng of total RNA using a miScript II RT Kit (Qiagen) per the manufacturer’s protocol. RT of pre-miR-200c and mRNAs was completed using qScript Kit (Quanta Bio) with 1000 ng of total RNA per the manufacturer’s protocol. qPCR was completed using a QS5 384-well PCR system (Applied Biosystems) using Power SYBR Green Master Mix according to the manufacturer’s protocol and the Comparative Ct with melt standard method, adjusted for a sample volume of 10 μL. All data were analyzed using the ΔΔCt method previously described (Rao et al., 2013 (link)).
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