Reactions were performed in triplicate on cDNA samples in 96-well optical plates on an ABI Prism StepOne System (ThermoFisher, U.K.). It consisted of 2 μL of cDNA, 7 μL of PCR-grade water, 10 μL of 2 × TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μL of Taqman specific assay probes (Applied Biosystems). Data were recorded using SDS software (v 2.3; PE Applied Biosystems). Advanced miRNA Taqman probes (ThermoFisher) were used for all miR qPCR assays.
Abi prism stepone system
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The ABI Prism StepOne System is a real-time PCR instrument designed for quantitative gene expression analysis and genotyping applications. It allows for the detection and quantification of nucleic acid sequences in a sample. The system includes a thermal cycler, optics for fluorescence detection, and software for data analysis.
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2 protocols using abi prism stepone system
1
Quantitative miRNA Expression Analysis
2
qPCR Assay for Gene Expression
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Reactions were performed in triplicate on cDNA samples in 96-well optical plates on an ABI Prism StepOne System (ThermoFisher). Reactions consisted of 2 μL cDNA, 7 μL PCR-grade water, 10 μL 2× TaqMan Universal PCR Master Mix (Applied Biosystems), 1 μL TaqMan-specific assay probes (Applied Biosystems) for PSA, and RPL19 or SYBR green primers for AR, AR-UTR (variants), β-actin (ACTB) and GAPDH (for primer details, see Supplementary Table 2, see section on supplementary materials given at the end of this article). Parameters were 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were recorded using Sequence Detector Software (SDS version 2.3; PE Applied Biosystems). Levels were normalised to GAPDH, β-actin and RPL19.
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