Absolute standard curve used to calculate the number of copies per pg of total RNA of each mRNA. The absolute standard curve was prepared with an in vitro synthesized RNA corresponding to a 457-nt fragment of the mouse Gapdh transcript (GenBank Database: M32599). Concentration was spectrophotometrically determined by A260 and converted to the number of copies using the molecular weight of the RNA fragment. Serial dilutions (109 to 102 RNA copies) were prepared, retrotranscribed, and amplified by real-time PCR. Primers have been previously described . The standard curve was constructed by plotting the log of starting RNA molecules versus the threshold cycle (Ct). The resulting standard curve is linear (r = 0.998) over 7 orders of magnitude. The efficiency (E) value is calculated from the slope of the standard curve equation, as E = 10[− 1/slope]− 1. The slope of the standard curve indicates that the standard is amplified with 100% efficiency. This standard curve was used to determine the number of copies of each experimental transcript, as exemplified for a Ct = 24.5.