Quantitative real-time polymerase chain reaction (qRT-PCR)

SA Sadaf Ashraf
MR Masar Radhi
PG Peter Gowler
JB James J. Burston
RG Raj D. Gandhi
GT Graeme J. Thorn
AP Anna M. Piccinini
DW David A. Walsh
VC Victoria Chapman
CM Cornelia H. de Moor
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500 ng of RNA was reversed transcribed to cDNA and diluted 5 fold with sterile distilled water before being subjected to qPCR using the GoTaq qPCR Master Mix containing the relevant forward and reverse primer sets (Supplementary Table 1). Primers for CD68, IL1β (spliced and unspliced), nestin, VEGF, PCNA, MYC, osterix, RUNX1, RUNX2 and TNF (spliced and unspliced) were designed with Primer Express 3.0 software. All qRT-PCR experiments were performed in triplicates, data were normalised to relative expression using the Qiagen Rotor-Gene Q software. All values were normalised to Ribosomal Protein L28 (RPL28) using the 2−∆∆ct method.

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