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Total cellular RNA was isolated from snap-frozen tissue by acidic phenol/chloroform extraction and DNase I treated (Roche Diagnostics, Mannheim, Germany). Then, 2 µg of RNA were reverse transcribed at 42 °C with 200 U of Moloney murine leukemia virus reverse transcriptase and 2 µM oligo d(T) 16 primer (Promega, Mannheim, Germany) in 25 µl of reaction mixture. Resulting cDNA was quantified by real-time polymerase chain reaction (RT-PCR) Master Mix (Applied Biosystems, Darmstadt, Germany) using FAM-5´ ⁄ TAMRA-3´ labeled probes for autophagy-related protein 3 (ATG-3), ATG-5, Beclin-1, Bcl-2 and HPRT (Metabion, Munich, Germany). Data represent the mean expression level ± standard deviation (standardized to HPRT expression) calculated according to the 2-∆∆CT method (13) of at least three independent measurements per cDNA (technical triplicates). The sequence of the primers used is listed in Table 1.. That aspect of treatment is usually performed in
That aspect of treatment is usually performed in . are not mechanically transmissible in nature. A recent fascinating.
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