Cells were lysed in TRI reagent (Sigma). RNA was then extracted with chloroform, precipitated with isopropanol and DNAse treated (DNA-free, Ambion). Total RNA (2 μg) was used per reverse transcription reaction. RNA was denatured in the presence of 2 μg of random hexamers (Invitrogen Life Technologies Ltd, Paisley, UK) for 5 minutes at 75°C, and reverse transcribed in a final volume of 40 μl with 200 U of SuperScript II (Invitrogen) at 42°C overnight followed by heat inactivation at 70°C for 15 minutes. Synthesized complementary DNAs were diluted in 300 μl of water and stored at −20°C until used.
Absolute quantification experiments were performed essentially as described in [39 (link)]. Briefly, 5.105 cells were lysed in 1 ml TRI reagent (Sigma) and 1 μl of a 1:20 dilution of ERCC Spike-in Mix#1 (Life Technologies Ltd, Paisley, UK) was added to each sample. After RNA extraction and reverse transcription, cDNAs were analysed by RT-qPCR. Transcript abundance were normalized to ERCC-00074 spike-in standard. ERCC-00096 and ERCC-00130 were also used as secondary controls.
Absolute quantification experiments were performed essentially as described in [39 (link)]. Briefly, 5.105 cells were lysed in 1 ml TRI reagent (Sigma) and 1 μl of a 1:20 dilution of ERCC Spike-in Mix#1 (Life Technologies Ltd, Paisley, UK) was added to each sample. After RNA extraction and reverse transcription, cDNAs were analysed by RT-qPCR. Transcript abundance were normalized to ERCC-00074 spike-in standard. ERCC-00096 and ERCC-00130 were also used as secondary controls.
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