Rneasy mini or midi kit

Total RNA was extracted using the RNeasy® Mini or Midi kit (Qiagen, Hilden, Germany) and digested with Rnase-free DNaseI (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The quality and quantity of RNA was assessed photometrically using the BioSpectrometer® (Eppendorf, Hamburg, Germany). To obtain cDNA, 1 μg of RNA was used for reverse transcription with a cDNA synthesis kit and random hexamer primers (Fermentas GmbH, St. Leon-Rot, Germany). Reverse-transcription polymerase chain reaction (RT-PCR) was performed using 1 μl cDNA using Taq DNA polymerase (JumpStart™ Taq DNA polymerase (Sigma-Aldrich) in a Mastercycler Gradient (Eppendorf, Hamburg, Germany) with primer pairs listed in Table S4 (Sigma-Aldrich). The RT-PCR conditions were as follows: 94°C for 2 minutes 30 seconds (initial denaturation), 25-35 cycles of 94°C for 30 seconds (denaturation), 60°C for 45 seconds (annealing), and 72°C for 60 seconds followed by 72°C for 5 minutes (final elongation). Samples were documented as described above.

Total RNA was extracted from tissue panels of the primates or mice transgenic animals using the RNeasy® Mini or Midi Kit from Qiagen. Tissues were homogenized using an OMNI rotor in a mixture of 300 μL buffer RLT plus 590 μL of RNase free water. The on-column DNase treatment was skipped, and total RNA was DNase treated with the Amnbion Turbo DNase kit. RNA quality and quantity were assessed by agarose gel electrophoresis. c-DNA were prepared using 1 μg of total RNA (Powersript™ Reverse Transcriptase from Clontech/Takara Bio kit and protocol). PCR reactions were performed using 1 μL of cDNA, Qiagen master mix, and primers specific for the ubiquitin-activating enzyme 1 (UBE1) and NPIP family (Additional file 2: Table S7). Cycling conditions consisted of 35 cycles with an annealing temperature of 55°. PCR products were run on a 1% agarose gel with 0.5 μg of 100 bp ladder.