Computational protocol: Adult diffuse gliomas produce mRNA transcripts encoding EGFR isoforms lacking a tyrosine kinase domain

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Protocol publication

[…] Complementary DNA (cDNA) was synthesized from 2 μg of total RNA using the Transcriptor First Strand cDNA Synthesis® kit (Roche) and hexamer primers, according to the manufacturer’s protocol. For PCR, primers were designed using Amplify 1.2 or Primer 3 (http://fokker.wi.mit.edu/primer3/input.html/) software and their specificity was determined by BLASTn in the NCBI database (http://www.ncbi.nlm.nih.gov/). Primer characteristics are listed in . Amplicon size and specificity were initially determined by 4% NuSieve agarose gel electrophoresis. Quantitative PCR [EGFRv1, -v2, -v3, -v4 and hypoxanthine phosphoribosyl transferase (HPRT)] and qualitative PCR (EGFRvIII) were performed on a Rotor Gene thermocycler (Corbett Research) using the Light Cycler Fast Start DNA Master SYBR Green I kit (Roche). All targets were amplified twice in duplicate in the presence of 3 mM MgCl2 and 0.5 μM primers. Relative quantification of mRNA content was performed using the ΔΔCt method [(Ctsample-Ctcalibrator)interest gene - (Ctsample-Ctcalibrator)reference gene], modified according to Pfaffl (), with efficiency correction by Rotor Gene software. mRNA content was expressed in relative arbitrary units (R.A.U.). […]

Pipeline specifications

Software tools Primer3, BLASTN
Application qPCR
Organisms Homo sapiens
Diseases Glioblastoma, Glioma, Neoplasms