Quantification of L1 RNA Abundance

Total RNA was isolated from transfected cell lys using TRIzol-LS (Invitrogen) and quantified (Nanodrop, Thermo Scientific). For qRTPCR analysis of transcript abundance, RNA (2 μg) was treated with RQ1 DNase (Promega) and cDNA synthesized from 1 µg using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). 2 µl cDNA were used for quantitative PCR using a TaqMan probe assay as described (5 (link)). For northern blots, total RNA and oligo dT-selected (poly A⁺, PureBiotech, MPG mRNA purification kit) RNA was fractionated through agarose formaldehyde gels, transferred to membrane and hybridized to ³²P-labeled in vitro transcribed antisense L1 RNA (NorthernMax Kit, Ambion). Phosphorimages were captured on a Typhoon 9400 and analysed with ImageQuant 5.2 (GE Healthcare). For RT-PCR to detect the AI, cDNA was synthesized using random hexamer from either 500 ng polyA+ or 2µg total RNA template in a 20 µl reaction, then 1 µl of that was amplified in a 25 µl PCR reaction using GFPint1F, 5′-GACTGGGTGCTCAGGTAGTGG and GFPint1R 5′-AAGATCCGCCACAACATCGA for 40 cycles. Products were separated on 2% agarose, stained with SYBRSafe (Invitrogen), and imaged on the Typhoon9400.