1. Quantitation of Hsp-72 mRNA in human muscle biopsies and heat-shocked NIH-3T3 cells, using competitive RT-PCR.
Quantitation of Hsp-72 mRNA in human muscle biopsies and heat-shocked NIH-3T3 cells, using competitive RT-PCR. A: Equal amounts of total RNA isolated from human muscles was added to titrated quantities (as indicated) of sRNA (competitor). RT-PCR of this sRNA yielded a product that was 80 nucleotides (nt) shorter (210 nt) than the amplification output generated from the native human Hsp-72 mRNA (290 nt, target), and that could therefore be distinguishable on the gel. B: The ratios of the amplification products from the competitor and target (C/T) were plotted against the number of copies of competitor added, and the plot was used to determine the equivalence point (indicated by arrows) between sRNA and target RNA. C: Kinetics of Hsp-72 expression after heat treatment of mammalian cells. NIH-3T3 cells were heat-treated at 43°C for 30 min and then incubated at 37°C. Total RNA was isolated at the indicated time points after heat treatment, and the amount of Hsp-72-specific message was determined with the competitive quantitative method. Results from two independent experiments (open or filled circles) are shown. Arrows indicate the beginning and the end of heat treatment. Basal Hsp-72 levels are shown by the two triangles. *106 copy/μg total RNA.
Istvan Kurucz et al. Diabetes 2002;51:
©2002 by American Diabetes Association