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Reverse transcription (RT) and real-time quantitative PCR (RQ-PCR)

DP Daina Pamedytyte
EL Enrika Leipute
BZ Birute Zilaitiene
VS Valdas Sarauskas
DD Dalia Dauksiene
AD Albertas Dauksa
AZ Aurelija Zvirbliene
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Reverse transcription and RQ-PCR were performed using the TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA), TaqMan miRNA assays (Applied Biosystems, Foster City, USA) and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, USA). Briefly, 20 ng of purified tumor-derived RNA was applied to RT and RQ-PCR was performed using Rotor-Gene 6000 PCR system (Corbett Research, Hilden, Germany) following the manufacturer ‘s protocols. The relative expression level was assessed using ΔΔCt method, where ΔCt is the difference in threshold cycles for target and reference genes. The fold expression changes were calculated by the 2−ΔCt method (Schmittgen and Livak 2008). Three RQ-PCR reactions were run per sample according to the manufacturer’s instructions. The limit of confidence for the Ct was 0.5.

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