Lightcycler 480 software release 1.5.0 sp3

Total RNA was isolated from cortex tissue of mice using miRNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA was synthesized from 1 μg of RNA using the SuperScript III Reverse Transcriptase (Life Technologies, Darmstadt, Germany), oligo-dT, and random N6 primers in a total volume of 20 μL. For quantitative reverse transcription-PCR (qPCR), 4 μl cDNA were used as template with 6 μl of Power SYBR Green PCR Master Mix (Life Technologies, Darmstadt, Germany) and 5 pmol of primers. The following primers were used: Ambra1 forward primer: 5′-AGG CTC CAG TGG TGG GAC TTC AC-3′, Ambra1 reverse primer: 5′-GCC AGG AGC TGA CCA TCT GCA G-3′, β-actin forward primer: 5′-CTT CCT CCC TGG AGA AGA GC-3′, β-actin reverse primer: 5′-ATG CCA CAG GAT TCC ATA CC-3′. qPCR reactions were run on LightCycler 480 System (Roche, Mannheim, Germany) with three technical replicates. Relative expression levels of Ambra1 were calculated using the threshold cycle method (LightCycler^® 480 Software release 1.5.0 SP3, Roche, Mannheim, Germany) and normalization to β-actin.

Total RNA was extracted with the Universal RNA Purification Kit (Roboklon, Berlin, Germany) and reverse transcribed. In total, 20 μl reactions with 50 ng of RNA, 1× RT Buffer, 1 U RNase inhibitor, 10 mM MgCl₂, random hexamers, 250 μM pooled dNTPs and 2.5 U MuLV reverse transcriptase (all products from Applied Biosystems, Foster City, CA) were prepared. The RT reaction was carried out in a T300 thermocycler (Biometra, Göttingen, Germany) for 15 min at 42 °C, with 5 min at 95 °C for deactivation of the enzyme and final cooling to 4 °C. qRT-PCR was performed in duplicate with the GoTaq qPCR Master Mix (Promega) in a final volume of 10 μl in a LightCycler 480 (Roche, Basel, Switzerland). PCR protocol included a pre-incubation step at 95 °C for 2 min, followed by 45 cycles of incubation at 95 °C for 7 s, annealing at 60 °C for 10 s and elongation at 72 °C for 5 s. In the melting curve, the temperature was increased from 65 °C to 95 °C (0.1 °C/s). Data were analysed with the LightCycler 480 Software release 1.5.0 SP3 (Roche). MACC1 or S100P expression was normalised to the expression of GAPDH. The following gene-specific primer were used: MACC1_fow 5’-ttcttttgattcctccggtga-3’; MACC1_rev 5’-actctgatgggcatgtgctg-3’; S100P_fow 5’-aatctagcaccatgacgg-3’; S100P_rev 5’-tctgcaggaagcctggta-3’; GAPDH_fow 5’-gaagatggtgatgggatttc-3’; GAPDH_rev 5’-gaaggtgaaggtcggagt-3’.

RNA isolation was performed using the Universal RNA extraction kit (Roboklon, Berlin, Germany). A quantity of 50 ng of total RNA was used for MuMLV-based cDNA synthesis (Biozym, Vienna, Austria). Primers for MACC1 were described elsewhere [1 (link)]. The primers for human IGFBP2 are forward: 5′-GCC CTC TGG AGC ACC TCT ACT-3′ and reverse: 5′-CAT CTT GCA CTG TTT GAG GTT GTA C-3′. All primers were synthesized at BioTeZ Berlin-Buch. qRT-PCR was performed as described earlier [3 (link)]. In brief, after initial denaturation at 95 °C, the amplification was performed for 40 cycles of denaturation (5 s; 95 °C) and a combined primer annealing and elongation step (45 s; 60 °C). Data were analyzed with LightCycler 480 Software release 1.5.0 SP3 (Roche Diagnostics, Penzberg, Germany). Mean values were calculated from duplicates. Each mean value of the expressed gene was normalized to its G6PDH level.