Faststart essential dna probes master mix

For RT-qPCR, mRNA of cultured cells was isolated and purified using Bioline Kit (Bioline, Meridian Bioscience, Luckenwalde, Germany), according to the manufacturer’s instructions, and stored at −80 °C for later use. The quality and concentration of mRNA were determined by measuring the absorbance at 260 nm and 280 nm on TECAN NanoQuant Plate^TM (Tecan Austria GmbH, Austria) using a spectrophotometer (Infinite 200 PRO, Tecan Austria GmbH) and calculating the absorbance ratio. The mRNA samples were treated with DNase (Promega, Walldorf, Germany) for 30 min at 37 °C to eliminate residual DNA contamination, and cDNA was synthesized from 1 µg of total mRNA in a volume of 40 µL using SensiFAST^TM cDNA synthesis Kit (Bioline, Meridian Bioscience, Luckenwalde, Germany) according to the manufacturer’s instructions. qPCR was performed using FastStart Essential DNA Probes Master Mix, primers (listed in the Supplemental Table S2), and the corresponding probes from Universal Probes Library on a Roche LightCycler^® 96 Instrument (all Roche Diagnostics GmbH, Mannheim, Germany). The amplification protocol was 10 min pre-incubation at 95 °C followed by 45 cycles of 10 s at 95 °C and 30 s at 60 °C. The expression levels of all genes were calculated using the 2^−(ΔCq) method relative to the average of 2 reference genes—β-Actin and β2-Microglobulin.

Total RNA was extracted from the hippocampus using the NucleoSpin RNA Kit (Takara, Otsu, Japan) according to the manufacturer’s instructions, and was then reverse transcribed into cDNA using Oligo-T priming and Moloney murine leukaemia virus reverse transcriptase (Takara). Quantitative polymerase chain reaction was performed using the FastStart Essential DNA Probes Master Mix (Roche, Basel, Switzerland) and run on a Step One Plus Real Time Polymerase Chain Reaction System using the Taqman gene expression assay mix (Applied Biosystems, Carlsbad, CA). Target gene mRNA expression was normalised to β-actin mRNA expression, and the relative amounts of all mRNAs were calculated using the comparative Ct method. The primer sequences and reaction parameters are shown in Table 1.

Human L1 copy number was measured with ORF2, HERVH or SATA as internal controls. The quantitative multiplexing PCR strategy to investigate endogenous L1 activity in humans was done as Gage et al. described12 (link). ORF2 probes were conjugated to the fluorophore label HEX and HERVH/SATA probes were conjugated with 6FAM. A 20-μl reaction volume containing 10 μl of 2 × FastStart Essential DNA Probes Master Mix (Roche, Cat. No. 06924492001), 1 μl of primers F/R (5 umol/L), 1 μl of probe (2.5 umol/L) and 1 μl of DNA (100 pg/μl) was used for quantitative PCR amplification. The detailed quantitative PCR conditions were as follows: 95 °C for 10 min, followed by 36 cycles of amplification and quantification (95 °C for 10 sec, 58 °C for 20 sec and 72 °C for 20 sec).