Computational protocol: A global microRNA screen identifies regulators of the ErbB receptor signaling network

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Protocol publication

[…] Total RNA was isolated from 5 × 105-1 × 106 cells using the mirVANA™ miRNA Isolation Kit (Life Technologies) according to the manufacturer’s protocol. Primers for ErbB3, RAP1A and GAPDH were QuantiTect Primer Assays from Qiagen, primers for SOS1 (forward: 5’-CGAGCCCTTTTCACTCAAGC; reverse: 5’-GCCATGGGGCAGAGTAACTT). were designed using PRIMER3 [,] and synthesized by biomers.net GmbH, Ulm, Germany. ErbB3, SOS1, RAP1A and GAPDH mRNA levels were quantified by qRT-PCR using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Foster City, CA, USA) according to manufacturer’s protocol. qRT-PCR was performed with a Cfx96 device (Biorad, Hercules, CA, USA). Changes in the relative expression level were calculated using the 2-ΔΔCt method (Biorad CFX manager software 2.1.). GAPDH was used as the endogenous control gene. […]

Pipeline specifications

Software tools Primer3, CFX Manager
Application qPCR
Diseases Breast Neoplasms, Neoplasms