(A) Quantitative real-time PCR (qRT-PCR) analysis of the levels of the long and short CIRP transcripts: Total RNA was isolated from NIH-3T3 cells cold shocked at 32°C for 2, 6, 12, and 24 h, or left to grow at 37°C for 24 h. The presence of the long (5′-UTR-L) and short (5′-UTR-S) 5′-UTRs, and the CIRP opening reading frame (ORF) transcript, was confirmed by nested PCR using specific primers. The presence of β-actin was detected as a control. The specific primers utilized are described in Table 1. Subsequently, total RNA was subjected to qRT-PCR using specific primers for the (B) CIRP ORF, (C) short 5′-UTR, and (D) long 5′-UTR and the relative levels of each message compared with the 37°C control determined. All data shown are the means of triplicate experiments ± SD.
