Total RNA was extracted from HORCs using the RNeasy Mini Kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. The concentration of total RNA was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA). Total RNA was reverse transcribed to complementary DNA (cDNA) in a reaction mix of Superscript II reverse transcriptase (Invitrogen, Paisley, UK), dNTP mix (Bioline, London, UK) and random primers (Promega, Southampton, UK) according to manufacturer instructions.
TaqMan PCR was performed using 5ng of input cDNA and Taqman PCR mastermix (Applied Biosystems, Warrington, UK) and human THY-1 primer and probe set (Hs00174816_m1, Assay on demand, Applied Biosystems, Warrington, UK). Amplification and detection was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Warrington, UK). THY-1 mRNA was normalised to the geometric mean of CT values for cytochrome c-1 (CYC-1) and topoisomerase DNA I (TOP1) as described previously [14 (link)]. Normalising genes were selected from a range of housekeeping genes using the Genorm protocol [17 (link)].
TaqMan PCR was performed using 5ng of input cDNA and Taqman PCR mastermix (Applied Biosystems, Warrington, UK) and human THY-1 primer and probe set (Hs00174816_m1, Assay on demand, Applied Biosystems, Warrington, UK). Amplification and detection was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Warrington, UK). THY-1 mRNA was normalised to the geometric mean of CT values for cytochrome c-1 (CYC-1) and topoisomerase DNA I (TOP1) as described previously [14 (link)]. Normalising genes were selected from a range of housekeeping genes using the Genorm protocol [17 (link)].
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