Rat tissues (10 mg each) were excised and stored at room temperature for up to 4 hours in phosphate-buffered saline (PBS) or RNAlater RNA Stabilization Reagent (RNAlater). At the indicated times, RNA was isolated using the RNeasy Protect Mini Kit. Total RNA was used for quantitative RT-PCR using the QuantiTect Probe RT-PCR Kit and primers and probe specific for the transforming growth factor β (TGF-β) gene or the tumor necrosis factor α (TNF-α) gene. Analyses were carried out in triplicate on the ABI PRISM 7700 Sequence Detection System. The change in threshold cycle (CT) is shown, relative to the CT at time zero.
RNA was isolated from fresh rat kidney samples after 0, 5, 10, 15, 30, and 60 minutes, using either standard procedures (Unstabilized) or RNeasy Protect Kits (Stabilized). The RNA isolated was analyzed by agarose gel electrophoresis and expression of GAPDH was examined using northern blot analysis. M: markers.
Total RNA was isolated from tissue samples stored in RNAlater RNA Stabilization Reagent. [A]Rat lung tissue was stored under the indicated conditions. RNA was analyzed on a formaldehyde agarose gel and northern blot (GAPDH-probed). [B]Stabilized mouse liver tissue was subjected to freeze–thaw cycles (–20°C/25°C). C: control tissues frozen in liquid nitrogen and stored at –80°C.