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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.

The synthesis of thymidylate synthase (TS) from 5-fluorouracil (FUra)- and 5-bromouracil (BrUra)-substituted mRNAs was examined to investigate the effect of incorporation of uracil (Ura) analogs on translation. Human TS cDNA was transcribed in the presence of Ura-, FUra-, or BrUTP to obtain 100% substituted mRNA. The mRNAs were translated in a rabbit reticulocyte lysate system. The TS protein that was formed from each of the templates reacted identically with TS antibody in Western blots. Time courses of TS formation revealed a characteristic peak which occurred at 45 min for the Ura- and FUra-RNAs and at 2 h for the BrUra-RNA. Substitution of Ura with FUra did not alter the rate of translation, while substitution of BrU for Ura decreased the rate of translation. Substitution of Ura with FUra or BrUra enhanced the stability of the mRNAs in the rabbit reticulocyte lysate by 3- and 10-fold, respectively. Incorporation of BrUra influenced the binding and catalysis on the ribosome, resulting in a 3.5-fold greater rate of activation (Kact) and 6-fold lower Vmax than the equivalent values for the Ura- and FUra-substituted mRNAs. Nondenaturing gel electrophoresis revealed that different conformations exist among the mRNAs. These data show that translation can be influenced by the incorporation of fraudulent bases into mRNA and those bases that stabilize RNA secondary structure will have the greatest inhibitory effect on translation.

The effects of mitomycin C (MMC) concentration and exposure time on the inhibition of tumor cell labeling index (LI) were studied using surgical bladder tumor samples from 14 patients. The bladder tumors were cultured as 1-mm3 fragments on collagen gels. LI was determined by incorporation of [3H]thymidine and autoradiography. All tumors responded to MMC. However, the sensitivity varied significantly between tumors. At a 2-h exposure, the concentrations required for 50 and 90% inhibition (IC50 and IC90) ranged from 0.237 to 14.9 and 2.76 to 74.5 micrograms/ml, respectively. There was an inverse correlation between MMC activity and tumor LI; the IC values were higher for the more rapidly proliferating tumors. Exposure time had a pronounced effect on MMC activity. Shortening the exposure time from 2 to 0.5 h increased the IC50 3-fold, while prolonging the exposure time from 2 to 24 h decreased the IC50 6-fold. To determine the minimum concentration and exposure time necessary to reduce tumor LI by 90%, the data for 6 tumors were computer fitted to the pharmacodynamic relationship Cn x T = k. The analysis showed that, on average, a 2.5-h exposure of 3 micrograms/ml was needed for 50% inhibition and a 7-h exposure of 8 micrograms/ml was needed for 90% inhibition. A comparison of the IC values of MMC determined in this study with the literature values determined using monolayer and spheroid cultures of established human bladder tumor cell lines showed that the drug activity in cultured tumor fragments ranged from 7- to 5300-fold lower than that in established cell lines. In summary, our data demonstrate a heterogeneity in the response of bladder tumors from individual patients to MMC, a decreased sensitivity to MMC with increasing tumor proliferation, and that drug concentration and exposure time are critical determinants of MMC activity.

Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named ‘hypoxia-induced noncoding ultraconserved transcripts' (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category.