Half-life and stability determination of β-actin and CIRP long and combined 5′-UTRs mRNAs at 37 and 32°C. Exponentially growing NIH-3T3 cells were cultured at 37 or 32°C for 24 h prior to the addition of actinomycin D (5 μg/mL). Subsequently, total RNA was isolated at 0, 2, 6, 12, and 24 h and the level of Cirp-5′UTR long, Cirp-5′UTR combined, and β-actin mRNA determined. (A) Exponential decay curves of β-actin mRNA at 37 (◆) and 32°C (▲), (B) decay curves of all three mRNAs at 37°C (actin ◆, combined 5′-UTR ■, long 5′-UTR ●), and (C) 32°C (actin ▲, combined 5′-UTR ■, long 5′-UTR ●). All data shown are the means of triplicate experiments ± SD. Half-life (t 1/2) was calculated by fitting two-parameter exponential decay curves as described in the Materials and Methods section. For further details see the text.
