The abundance of RNAs encoding cholesterol import (LDLR) and cholesterol synthesis-regulating (HMG-CoAR, SS) proteins are substantially increased by mevastatin treatments in NB4 AML cells. NB4 cells were incubated for 18 to 24 hours with 12.5 μM mevastatin, total cellular mRNAs were prepared from untreated and treated cells, and oligo(dT)–primed cDNAs were synthesized. For semiquantitative RT-PCR, LDLR intron-spanning primer pairs were multiplexed with GAPDH primers using conditions optimized to ensure amplification in the linear range for both products, as determined by image analysis of ethidium bromide-stained agarose gels (A-B). Arrows indicate 3-μL cDNA inputs. (C) For real-time RT-PCR, β₂-microglobulin DNA and NB4 cell line RNAs were serially diluted into yeast RNA and processed to create standard curves. Quenched, fluorescent internal probes allowed the amount of PCR amplicon created to be quantitated per cycle as fluorescence on an ABI Prism 7700 detector. Standard curves were generated for β₂-microglobulin using plasmid DNA and for β₂-microglobulin, LDLR, and HMG-CoAR using the NB4 cell line. Standard curves are shown that plot the cycle number at which signals were detected above threshold (y-axis) against the starting quantities of β₂-microglobulin plasmid (10-10⁸ copies/μL) diluted into yeast RNA (x-axis) or against the starting quantity of NB4 cell RNA (0.01-100 ng/μL). (D) Methods similar to those used in panels A and B were used to optimize HMG-CoAR and SS RT-PCRs, as described more completely in “Materials and methods.” When standardized to GAPDH signals and normalized to untreated signals, mean LDLR and HMG-CoAR signals were significantly increased in mevastatin-treated samples (5 independent assays), and mean SS signals (n = 3) were also increased. (E) RNA quantities were normalized based on β₂-microglobulin levels in real-time PCR, as shown in panel C, and LDLR and HMG-CoAR mRNA levels in mevastatin-treated and untreated NB4 cells are expressed relative to the untreated NB4 cell line controls. Means are plotted in panels C and E, and error bars represent SEM. Wilcoxon rank sum tests were used to compare means of treated and untreated AML samples along with 2-tailed tests of significance.