Includes step-by-step instructions, a free template, and real-life examples.
A total of 5 pmoles of forward and reverse primer, cDNA template, and Real-Time 2× PCR Master Mix SYBR A (A&A Biotechnology, Gdynia, Poland) were used.
Three hundred nanograms of total RNA was used as a template and real-time RT PCRs were performed with MiniOpticonTM Version 3.1(Bio-Rad, Hercules, CA,USA) in combination with the QuantiTect SYBR Green RT-PCR (Qiagen GmbH, Hilden, Germany).
The algorithm consists of three steps: noise analysis, template generation and real time detection and classification.
cDNA was used as a template, and quantitative real-time RT-PCR was performed with Bio-rad 170-9780 iQ5 Multicolor Real-Time PCR Detection System (BioRad, USA) according to the manufacturer's protocol.
FP assisted NO with DNA template selection and real-time primer design.
All reverse transcriptase reactions included no-template controls and real-time minus controls.
All real-time reactions, including no-template controls and real-time minus controls, were run in a GeneAmp PCR 9700 thermocycler (Applied Biosystems).
MiRNA and mRNA quantitative RT PCR, including no-template controls and real-time minus controls, were performed in triplicate and run in a CFX 384 Touch Real-Time PCR Detection System (Bio-Rad, Munich, Germany).
qPCR was performed with 10 ng RT template and SYBR Premix ExTaq (Perfect Real Time, Takara, Japan), using the Mini Opticon Real-Time PCR system (BioRad, USA).
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