Amplification and Characterization of HERV-K Transcripts

We amplified rec and np9 mRNA representing PCR products from tissue cDNA panels employing forward primers rec-np9-for-1: 5′-ATG AAC CCA TCA GAG ATG CAA-3′; rec-np9-for-2: 5′-ATG AAT CCA TCA GAG ATG CAA-3′; rec-np9-for-3: 5′-GCG AAC CCT TCA GAG ATG CAA-3′; rec-np9-for-4: 5′-ATG AAC CCA TCG GAG ATG AAA-3′; that were combined in a ratio of 85/5/5/5, and reverse primers rec-np9-rev-1: 5′-AGC ATC TGT TTA ACA AAG CA-3′; rec-np9-rev-2: 5′-AGC ATG TTT AAC AAA GCA-3′ 5% combined in a ratio of 95/5. The various primer variants considered sequence differences of HERV-K(HML-2) loci within primer binding regions. PCR products were amplified using standard conditions with AmpliTaq Gold (Applied Biosystems/Life Technologies, Carlsbad, CA, USA) DNA polymerase and the following PCR program: 12 min 95°C; 35 cycles: 50 s 95°C, 50 s 58°C, 30 s 72°C, and final elongation 10 min 72°C. PCR products were separated by agarose gel electrophoresis; np9 and rec representing PCR products were purified from gels using NucleoSpin Gel and PCR Clean-Up Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany). Products were cloned into pCR II-TOPO (Invitrogen/Life Technologies) and transformed into Escherichia coli DH-5α cells. Plasmid DNA from randomly selected bacterial colonies was purified and subjected to Sanger sequencing (see below).
We amplified rec and np9 mRNA representing cDNA from total RNA from heart, brain, and colon tissues by RT-PCR following a previously established procedure [21 (link)]. PCR products were cloned into pGEM T-Easy (Promega GmbH, Mannheim, Germany). Plasmid DNA from randomly selected bacterial colonies was prepared as described before [32 (link)].
cDNA inserts were sequenced using vector-specific T7 primer and an Applied Biosystems 3730 DNA-Analyzer (Seq-IT GmbH, Kaiserslautern, Germany). Sequence qualities were verified by eye, and poor quality sequence reads were excluded from further analysis.