Quantification of CD44 Expression by RT-qPCR

Total RNA was isolated using Trizol (Sigma-Aldrich, Taufkirchen, Germany), complementary DNA was synthesized using Ready-To-Go-You-Prime-First-Strand Beads (GE Healthcare (VWR), Bruchsal, Germany) and RT-qPCR performed using GoTaq RT-qPCR Master Mix (Promega, Mannheim, Germany) and Eppendorf fluorescence thermocyclers (Eppendorf, Wesseling-Berzdorf, Germany), all following manufacturers' instructions. The 2^ΔΔCT method was used to quantify amplified fragments. Expression levels were normalized using at least one housekeeping gene (rps29 and/or rpl4). The sequence of the qPCR primers can be found in ref. 47 (link). The sequences of the primers used for CD44 are: 5′-GCACTGTGACTCATGGATCC-3′ present in exon 16 and 5′-GCACTGTGACTCATGGATCC-3′ in exons 17–18.