Differences between stabilization and activation. (A) Stabilization: relative activity of SfyI at various temperatures in the absence or presence of trehalose. (B) Activation: relative activity of DNaseI and NcoI restriction enzyme at the indicated temperatures in the absence or presence of trehalose. °C axis, temperature of incubation; % axis, relative activity (arbitrary units). Data are representative of three independent sets of experiments.

Comparison of trehalose with other sugars for enzyme stabilization or activation. In vitro transcript of 5 kb was used as substrate for cDNA synthesis at 60°C in the presence of the following reaction modifiers: lane 1, trehalose; lane 2, sucrose; lane 3, glucose; lane 4, maltose; lane 5, no addition. Arrow and asterisk, longest full length cDNA detectable in the trehalose-containing reaction and in the absence of trehalose, respectively. Data are representative of more than three independent experiments.

Improvement of full length cDNA yield by thermostabilization of RT by trehalose. (A) 5-kb cDNAs were synthesized in the presence of 0.6 M trehalose at 45°C (lane 1), 55°C (lane 2), 60°C (lane 3), and 65°C (lane 5); lane 4, synthesis at 60°C in the absence of trehalose. (B) In vitro transcripts were used as templates as follows: lanes 1–4, 5 kb; lanes 5–8: 5.5 kb; lanes 9–12: 10 kb. Lanes 1, 5, and 9: reactions without trehalose; lanes 2–4, 6–8, and 10–12: reactions with trehalose. Lanes 1, 2, 5, 6, 9, and 10: incubation at 45°C; lanes 3, 7, and 11: incubation at 60°C; lanes 4, 8, and 12: incubation with temperature cycling. Arrowheads, full length cDNAs; asterisks, truncated cDNAs. (C) RT of mouse brain mRNA: lane 1, standard optimized reaction; lane 2, reaction in the presence of trehalose at standard temperature; lane 3, reaction in absence of trehalose with the temperature-cycling program; lane 4, reaction in presence of trehalose with the temperature cycling program. M, λ-HindIII markers. Figures are representative of at least three independent experiments. Longer cDNAs synthesized in the presence of trehalose (lane 4, arrow) or under standard best conditions (lane 1, asterisks) are shown.