Mircury lna exilent sybr green master mix

RNA for profiling of miRNA was isolated from FFPE tissue using the RecoverAll™ total nucleic acid isolation kit (Applied Biosystems, Carlsbad, California, USA). RNA concentrations were quantified using the NanoDrop Spectrophotometer (Nanodrop Technologies, Montchanin, New Castle, Delaware, USA). Afterwards, reverse transcription (RT) using amounts of 20 ng of total RNA by applying the miRCURY LNA™ Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark), containing synthetic RNA Spike Ins, was performed. 5 μl of the RT products was combined with the PCR master mix and nuclease-free water from the miRCURY LNA™ ExiLENT SYBR® Green master mix (Exiqon, Vedbaek, Denmark). After that, 10 μl of the PCR Master mix-cDNA mix was added to each 384-well plate of the miRCURY LNA™ Universal RT miR Ready-to-Use PCR, Cancer focus panel, V4 (Exiqon, Vedbaek, Denmark). Finally, qPCR was performed by using the LightCycler® 480 instrument (Roche molecular systems Inc., Mannheim, Germany). All reactions were carried out according to the manufacturer's instructions. Initial data analysis was executed by using the LightCycler® 480 Software (Roche molecular systems Inc., Mannheim, Germany) to obtain raw Ct values. Ct values were used to determine the amount of miRNA in a sample (both parameters showing an inverse correlation).