Targetamp 1 round aminoallyl arna amplification kit

RNA from each biological replicate (pool of eight individuals) was amplified (TargetAmp™ 1-Round Aminoallyl-aRNA Amplification Kit, Epicentre Technologies Corporation) according to the manufacturer’s instructions. Following quality control (Nanodrop quantification and agarose gel electrophoresis), amplified RNA fragments (aRNA) were indirectly fluorescently labelled and purified. Briefly, dye suspensions (Cy3 and Cy5) in sufficient quantity for all labelling reactions were prepared by adding 42 µL of high-purity dimethyl sulfoxide (Stratagene) per tube of Cy dye (PA23001 or PA25001; GE HealthCare). Individual amplified samples (2.5 µL aRNA in 10.5 µL H₂O) were denatured at 75 °C for 5 min, and then 3 µL 0.5 M NaHCO₃ at pH 8.5 and 1.5 µL Cy3 dye were added. The common reference pool was labelled in the same way, but it was prepared in a single large-scale reaction i.e. 50 µg of pooled aRNA in 210 µL H₂O were heat-denatured and then 60 µL 0.5 M NaHCO₃ at pH8.5 and 20 µL Cy5 dye were added. All samples were incubated for 1 h at 25 °C in the dark, and purified through Illustra AutoSeq G-50 Dye Terminator columns (Qiagen). Dye incorporation and purity of all reactions were assessed spectrophotometrically (NanoDrop) and the products were also visualised on a fluorescent scanner (Typhoon Trio, GE Healthcare).

Each pooled RNA sample was amplified (TargetAmp™ 1-Round Aminoallyl-aRNA Amplification Kit, Epicentre Technologies Corporation, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Following quality control (Nanodrop quantification and agarose gel electrophoresis) each aRNA sample was indirectly labelled and purified. Briefly, Cy dye suspensions (Cy3 and Cy5) in sufficient quantity for all labelling reactions were prepared by adding 40 μL high purity dimethyl sulphoxide (Stratagene, Hogehilweg, The Netherlands) per tube of Cy dye (PA23001 or PA25001; GE HealthCare, Little Chalfont, Bucks, UK). Each sample (2.5 μg aRNA) was denatured at 75°C for 5 min and then 3 μL 0.5 M NaHCO₃ pH8.5 and 1.5 μL Cy3 or 1.0 μL Cy5 dye was added achieving a total volume of 15 μL per reaction. Samples were incubated for an hour at 25°C in the dark, purified using Illustra AutoSeq G-50 Dye Terminator Removal Kit (Qiagen GE Healthcare) and concentration, dye incorporation and purity were assessed via spectrophotometer (NanoDrop) with products also visualised on a fluorescent scanner (Typhoon Trio, GE Healthcare).

Each pooled RNA sample was amplified (TargetAmp 1-Round Aminoallyl-aRNA Amplification Kit, Epicentre Technologies Corporation, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Following quality control (Nanodrop quantification and agarose gel electrophoresis) each aRNA sample was indirectly labelled and purified. Briefly, Cy dye suspensions (Cy3 and Cy5) in sufficient quantity for all labelling reactions were prepared by adding 42 μL high purity dimethyl sulphoxide (Stratagene, Hogehilweg, The Netherlands) per tube of Cy dye (PA23001 or PA25001; GE HealthCare, Little Chalfont, Bucks, UK). Individual samples (2.5 μg aRNA in 10.5 μL H₂O) were denatured at 75 °C for 5 min and then 3 μL 0.5 M NaHCO₃ pH 8.5 and 1.5 μL Cy3 dye added. The reference pool consisted of the same proportions per sample, but 1 μL Cy5 dye was used to label 2.5 μg pooled aRNA. Samples were incubated for an hour at 25 °C in the dark, purified using an Illustra AutoSeq G-50 Dye Terminator Removal Kit (Qiagen GE Healthcare), and concentration, dye incorporation and purity were assessed via spectrophotometer (NanoDrop) with products also visualised on a fluorescent scanner (Typhoon Trio, GE Healthcare).