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 effects of serum on the expression of four commonly used housekeeping genes were examined in serum-stimulated fibroblasts in order to validate the internal control genes for a quantitative RT-PCR assay. NIH 3T3 fibroblasts transfected with an inducible chimeric gene were serum-starved for 24 h and then induced with 15% serum for 8 h. Serum did not alter the amount of total RNA that was expressed in the cells, however, the amount of mRNA significantly increased over time with serum-stimulation. Both messenger and total RNA from each of the time points were reverse transcribed under two different conditions; one in which the reactions were normalized to contain equal amounts of RNA and another series of reactions that were not normalized to RNA content. The resulting cDNA was amplified by real-time, quantitative PCR using gene-specific primers for beta-actin, beta-2 microglobulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA. The expression of beta-actin and GAPDH increased up to nine- and three-fold, respectively, under all conditions of reverse transcription (P<0.01). The expression of 18S rRNA increased with serum-stimulation when the cDNA synthesized from non-normalized, total RNA was assayed (P<0. 01) but not when the reverse transcriptions were normalized to RNA content (P>0.05). The expression of beta-2 microglobulin increased up to two-fold when assayed from cDNA synthesized from non-normalized mRNA, but was unaffected by serum when the reverse transcriptions were normalized to mRNA. beta-2 Microglobulin expression was found to be directly proportional to the amount of mRNA that was present in non-normalized reverse transcription reactions. Thus, beta-2 microglobulin and 18S rRNA are suitable internal control genes in quantitative serum-stimulation studies, while beta-actin and GAPDH are not. The internal control gene needs to be properly validated when designing quantitative gene expression studies.

Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3'-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to beta-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r(2) obtained from the linear regression of the first-order mRNA decay plots was SYBR green>> TaqMan>> probe hybridization>> band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.

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.

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.

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.

Freshly isolated suspensions of rat parenchymal liver cells (hepatocytes) produce large amounts of nitrite following isolation. Nitrite production was inhibited by the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine, as well as the transcription inhibitor actinomycin D. Increases in iNOS mRNA, protein, and activity levels correlated with the formation of nitrite. iNOS mRNA was first detectable 2 h after the onset of hepatocyte incubations and peaked at 4 h. These results indicate that nitrite formation is a result of the large scale production of nitric oxide (NO) by hepatocytes in response to the time-dependent induction of iNOS. NO production by hepatocytes was attenuated by pretreatment of rats with the Kupffer cell inhibitor, gadolinium chloride. Also, the addition of the endotoxin neutralizing agent, polymyxin B; the protein kinase inhibitor, staurosporine, and antioxidants to perfusion buffers and hepatocyte suspensions also decreased nitrite formation. Collectively, our results suggest that iNOS is induced in hepatocytes in response to the stresses generated during collagenase isolation procedures. The response appears to be triggered by a complex interaction between several different factors including Kupffer cell activation, reactive oxygen species generation, and endotoxin contamination of collagenase preparations.

Freshly isolated suspensions of rat parenchymal liver cells (hepatocytes) spontaneously produce large amounts of nitrite following collagenase isolation. Our previous studies indicate that nitrite production is associated with the expression of inducible nitric oxide synthase (iNOS) and reflects NO production. Depletion of glutathione (GSH) with diethylmaleate (DEM) inhibited nitrite production, and this inhibition was time-dependent. DEM was more effective in blocking nitrite production if it was added within the first 1 hr of the start of the incubation. The reducing agent dithiothreitol (DTT) and the alkylating agent ethyl methanesulfonate (EMS) also inhibited hepatocyte nitrite production, and this inhibition was also greatest if they were added within 1 hr of initiating the incubation. However, EMS added at 3 hr still reduced 6-hr nitrite production by about 70%. This reduction in nitrite production by EMS added at 3 hr may be due to the direct modification of thiol groups on the iNOS protein because we have determined that iNOS activity is inhibited by the sulfhydryl modifying reagent N-ethylmaleimide (NEM). Western blots also indicate that the iNOS protein is expressed when EMS is added at 3 hr. The addition of DEM, DTT, or EMS at 0 time greatly reduced the levels of cellular iNOS mRNA relative to controls as determined by quantitative RT-PCR. Based on our results with mRNA levels, both DTT and depletion of cellular GSH appear to inhibit the early signaling events leading to iNOS expression and suggest that the control of iNOS induction in hepatocytes is sensitive to the thiol redox status of the cell.

This study examined the in vitro proliferation and mitomycin C (MMC) sensitivity of patient bladder tumors as a function of the tumor pathobiology. Surgical specimens of transitional cell carcinoma of the bladder were maintained as histocultures on collagen gels. The thymidine labeling index (LI) was determined by autoradiography and the labeling for proliferating cell nuclear antigen (PCNA) by immunohistochemistry. There was a linear correlation between the thymidine LI and the PCNA LI, but the PCNA LI were quantitatively lower than the thymidine LI. The mean thymidine LI were 30.9, 32.4 and 51.5% for the grade I, II and III tumors, 33.6 and 56.3% for the superficial (Tis, Ta and T1) and invasive (T2-T4) tumors, and 28.9 and 50.9% for the diploid and aneuploid tumors. Analysis of variance indicates that these differences were statistically significant. These data indicate that the proliferation of tumor histocultures paralleled the tumor aggressiveness in vivo. The tumor sensitivity to MMC, measured by the inhibition of the thymidine LI of tumor cells, was studied in 31 tumors. At a 2 hour exposure, as is currently used in intravesical therapy, the MMC concentrations required for 50% inhibition of thymidine LI (IC50) showed a 120-fold intertumor variation (0.102 to 12.4 micrograms./ml.). The sensitivity to MMC inversely correlated with tumor aggressiveness. The IC50 increased with tumor LI (p < 0.05). The mean IC50 were 2.61 and 5.79 micrograms./ml. for superficial and invasive tumors (p < 0.05), 1.06, 3.05 and 4.49 micrograms./ml. for grade I, II and III tumors (p < 0.05), and 2.53 and 4.31 micrograms./ml. for diploid and aneuploid tumors (p = 0.14). These data indicate a large difference in sensitivity of human bladder tumors to MMC, with greater sensitivity for well-differentiated superficial tumors and lesser sensitivity for undifferentiated, invasive tumors.

Human bladder tumor fragments were cultured on collagen gel. In this system, the three dimensional architecture, cell-to-stroma and cell-to-cell interactions, and tumor heterogeneity were maintained. Cell viability and labeling index (LI) were determined by exposure to 3H-thymidine and autoradiography. Of the samples from 20 patients with transitional cell carcinoma, 14 (70%) were successfully cultured and had a mean LI of 32%. In addition, one specimen from a patient with squamous cell carcinoma was cultured and had a LI of 61%. Cultured samples were tested for chemosensitivity using a two hour exposure of mitomycin C in concentrations ranging from one to 50 micrograms./ml. A dose-dependent relationship was demonstrated; LI decreased as mitomycin C concentrations increased. The methodology described provides an alternative to suspension or monolayer techniques of culturing human bladder tumors for pharmacological studies.

In a previous study, we showed that 5-fluorouracil (FU) is active against the dimethylhydrazine-induced colon tumor in rats; a 7-day infusion of FU at 30 mg/kg daily produced 85% tumor-free cures. The present study examined the effects of FU alone and in combination with leucovorin (LV) or D-glucarate (GT) using an ex vivo system that maintained the growth of the rat colon-tumor explants on collagen gels. The labeling index (LI) was determined by the incorporation of [3H]-thymidine and autoradiography. The mean LI of the untreated control was 64.8% +/- 19.8%. The IC50, IC90, and IC95 values following a 7-day exposure to FU were 0.36, 0.75, and 1.22 microM, respectively. In comparison, the steady-state FU concentration required to produce 67% tumor-free cures in rats following a 7-day infusion is 1.54 microM. LV alone did not produce any antiproliferative effect at concentrations as high as 10 microM. The addition of LV at concentrations of 0.001-10 microM did not significantly reduce the IC50 of FU. The lack of effect of LV may have been due to tissue saturation with folate provided in the culture medium. GT alone reduced the tumor LI by 20%-30% at concentrations of 0.1-10 microM. GT enhanced the effect of FU. As compared with FU alone, the addition of GT at concentrations of 0.1 and 1.0 microM reduced the IC50 of FU by 47% and 60% to 0.21 and 0.16 microM, respectively. Assessment of the potentiation of the inhibitory effect of FU by GT using two-way analysis of variance and the isobologram method indicated a significant synergistic interaction between FU and GT. This interaction occurred within the FU concentration range of 0.08 and 0.4 microM. In summary, these data indicate that (a) the IC values for FU are comparable in tumor explants and in rats, suggesting that the effects in cultured tumors reflect those in intact animals; (b) GT alone showed antitumor activity, albeit relatively minor as compared with FU; (c) FU and GT exhibited synergistic activity, which was most pronounced at FU concentrations that produced submaximal activity (less than 30% inhibition of tumor LI); and (d) GT and LV had different effects on the growth inhibition by FU, suggesting that GT acts by a mechanism different from the thymidylate synthase-directed effect of FU and LV.