The only minus point: because they open at 5pm, they will only screen the 8pm matches.
Four of these enzymes were engineered with RNAse H minus point mutations to render them non-degrading to RNA, while one had RNase H activity substantially reduced, in addition to other characteristics and optimizations (see Tables 1 & S1 for more information).
For mRNA analysis cDNA was randomly primed from 1 μg of total RNA following DNase 1 treatment (New England Labs) using M-MLV reverse transcriptase RNase H minus, point mutant (Promega) and Random primer 6 (New England Labs).
Total RNA was treated with DNAse (RQ1, RNAse free, Promega Catalys, Wallisellen, Switzerland), followed by M-MLV reverse transcriptase (RNAse H minus, point mutant, Promega, Catalys) according to the manufacturer's recommendation and stored at −20°C.
After dosage using NanoDrop (witec ag ND-1000 Spectrophotometer) and quality control on 1% agarose gel, 1.5 µg of RNA were treated by RQ1 RNase-free DNase and retro-transcribed using M-MLV Reverse Transcriptase RNase H Minus Point Mutant (Promega) following the datasheet protocol.
For assessment of c-FLIP mRNA expression, total RNA was obtained from cell pellets using Qiagen RNeasy mini kits (Qiagen, Doncaster, VIC, Australia) and reverse transcribed using M-MLV Reverse transcriptase (RNase H Minus, Point mutant) and random primers (Promega, Madison, WI, USA).
