Figure 2.

Quantitative RT-PCR analyses of mRNA for aggrecan (A) and type II collagen (Col2a1) (B) in the cmd mice. mRNA was prepared from cartilaginous tissue of day 15 embryonic limbs of cmd homozygote, cmd heterozygote, and wild-type mice. Quantitative RT-PCR analysis was performed using the PCR MIMIC Construction kit (CLONTECH). The RT-PCR mixture contained a constant amount of cDNA, various amounts of the neutral mimic DNA as a competitor, and two oligonucleotide primers for aggrecan or Col2a1. The competitor DNA fragment (mimic DNA) was unrelated to the aggrecan gene or to Col2a1 except for containing the same short sequences for aggrecan or Col2a1 at both ends. mRNA levels for wild-type mice (Top), cmd heterozygotes (Middle), and cmd homozygotes (Bottom) were determined. Mimic DNA added was 10 or 80 attomoles (amol) (1 amol = 10⁻¹⁸ mol) (lane 1); 5 or 40 amol (lane 2); 2.5 or 20 amol (lane 3); 1.25 or 10 amol (lane 4); 0.63 or 5 amol (lane 5); 0.31 or 2.5 amol (lane 6); and 0.15 or 1.5 amol (lane 7) for aggrecan or Col2a1, respectively. Open arrowheads indicate mimic PCR product. Closed arrowheads indicate PCR product for aggrecan or Col2a. (Lower graphs) Ratios of cDNA for aggrecan or Col2a1 to PCR mimic vs. input mimic DNA amounts are shown.