QuantiNova SYBR Green PCR Kit (2500)

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Publication protocol

mRNA testing: Total RNA was extracted from approximately 30 mg cancer and non-neoplastic tissues using a TRIzol Plus RNA Purification Kit (Life Technologies, Mulgrave, Australia) as per manufacturer’s recommendation (one tissue sample was not available for analysis). The concentration of RNA was estimated by measuring the absorbance at 260 nm (NanoDrop 8000 UV-Vis Spectrophotometer). For complementary DNA synthesis, 1 μg of RNA diluted in nuclease-free water was reverse-transcribed using a QuantiNova Reverse Transcription Kit (Qiagen). The reaction was performed at 25°C for 3 minutes, followed by 10 minutes at 45°C, whereafter the reaction was terminated by heating at 85°C for 5 minutes. The mRNA levels of BCAT1, IKZF1, and HPRT1 (reference gene) were assessed in triplicate using a QuantiNova SYBR Green PCR Kit (Qiagen) and real-time PCR (Rotor-Gene Q instrument). Primers for the amplification of BCAT1 were designed by Ensembl database (http://asia.ensembl.org/index.html), whereas IKZF1 and HPRT1 primers sets were obtained from published papers (Table 1).25,26 Normalization was performed using Q-Gene application,27 with the expression of BCAT1 and IKZF1 relative to HPRT1 expressed as the mean normalized expression.

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