title: VG-CARE_Homo sapiens_20 gene expression panel for hepatocellular carcinoma
status: Public on Dec 25 2021
contact.city: Tuebingen
contact.first: Srinivas
contact.middle: Reddy
contact.last: Pallerla
contact.email: thecnu@googlemail.com
contact.state: Baden-Württemberg
contact.address: Wilhelmstraße 27
contact.department: Institute for Tropical Medicine
contact.country: Germany
contact.institute: Universitätsklinikum Tübingen
contact.phone: 004970712982197
organism: Homo sapiens
description: HCC PCR assay panel
  For cDNA synthesis, one microgram of total RNA from each sample was reverse transcribed in 20 µL reaction using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The cDNA was diluted to 1:10 with nuclease-free water and subjected to quantitative real-time PCR. The quantitative real-time PCR was performed with the LightCycler 480 Instrument II (Roche, Basel, Switzerland) in 96 well plates using a 10 µL final volume containing 0.3 µmol/L of forward and reverse primers, 2 µL of diluted cDNA and 1X SensiFAST™ SYBR® No-ROX Kit (Bioline, Memphis, TN, USA). PCR cycle parameters were initial denaturation at 94 °C for 2 min, 40 cycles at 95 °C for 10 s, and 60°C for 25 s, with the fluorescence signal acquired at the end of each cycle. A melting curve analysis was performed at the end of the amplification. All 23 genes (20 genes under investigation plus 3 housekeeping genes) were quantified in duplicates in 96 well plates having a couple of non-template reactions with ACTB primers. The primer sequences used are given in the supplementary table.
manufacture_protocol: Primers details used in the panel and relevant citations are in the readme file attached.