BACKGROUND:Quantitative polymerase chain reaction (qPCR) is the technique of choice for quantifying gene expression. While the technique itself is well established, approaches for the analysis of qPCR data continue to improve.RESULTS:Here we expand on the common base method to develop procedures for testing linear relationships between gene expression and either a measured dependent variable, independent variable, or expression of another gene. We further develop functions relating variables to a relative expression value and develop calculations for determination of associated confidence intervals.CONCLUSIONS:Traditional qPCR analysis methods typically rely on paired designs. The common base method does not require such pairing of samples. It is therefore applicable to other designs within the general linear model such as linear regression and analysis of covariance. The methodology presented here is also simple enough to be performed using basic spreadsheet software.

Gametophytes of the fern Ceratopteris richardii develop into either hermaphrodites or males. As hermaphrodites develop, they secrete antheridiogen, or ACE, into the environment, inducing male development in undifferentiated gametophytes. Hermaphrodites are composed of archegonia, antheridia, rhizoids and a notch meristem, while males consist of antheridia and rhizoids. Much of the research on sexual and morphological development concerns gametophytes grown in sterile environments. Using biochemical and molecular techniques we identify a soil bacterium and explore its effects on sexual and rhizoid development. Hermaphrodite and male gametophytes were exposed to this bacterium and the effects on sexual development, rhizoid length and rhizoid number were explored. The bacterium was identified as a pseudomonad, Pseudomonas nitroreducens. Gametophytes grown in the presence of the pseudomonad were more likely to develop into hermaphrodites across all gametophyte densities. Across all gametophyte sizes, hermaphrodites had rhizoids that were 2.95× longer in the presence of the pseudomonad while males had rhizoids that were 2.72× longer in the presence of the pseudomonad. Both hermaphrodite and male gametophytes developed fewer rhizoids in the presence of the pseudomonad. Control hermaphrodites produced 1.23× more rhizoids across all gametophyte sizes. For male gametophytes grown in the absence of the pseudomonad, the rate of increase in the number of rhizoids was greater with increasing size in the control than the rate of increase in males grown in the presence of the pseudomonad. The pseudomonad may be acting on gametophyte sexual development via several potential mechanisms: degradation of ACE, changes in nutrient availability or phytohormone production. The pseudomonad may also increase rhizoid number through production of phytohormones or changes in nutrient availability.

qPCR has established itself as the technique of choice for the quantification of gene expression. Procedures for conducting qPCR have received significant attention; however, more rigorous approaches to the statistical analysis of qPCR data are needed.

The development of male Ceratopteris richardii Brongn. gametophytes (induction) is well studied. Males develop in response to hermaphrodite-produced antheridiogen, which coincides with induced expression of ANI1 (antheridiogen-induced 1). Induction is blocked by exogenous abscisic acid (ABA). Conversion of male gametophytes to hermaphrodites occurs when males are removed from antheridiogen and is a less understood process. In this study, males were exposed to ABA at 0 μmol·L−1, 1 μmol·L−1, 10 μmol·L−1, 100 μmol·L−1, and 300 μmol·L−1and monitored for conversion. RT-qPCR methodology was developed to examine ANI1 expression levels in gametophytes outside of induction. Conversion of male gametophytes occurred more often and more quickly in media lacking ABA, and at lower ABA concentrations than at higher concentrations. ANI1 expression dropped significantly in males transferred to media lacking antheridiogen, but remained high in males remaining in the presence of antheridiogen and in those transferred to ABA. ANI1 expression was higher in 24 day old hermaphrodites relative to 24 day old males and at comparable expression levels to 6 day old gametophytes. These results suggest ABA serves a distinct role in conversion compared with induction, and ANI1 is expressed throughout the male’s lifespan and in older hermaphrodites.

For annuals, the timing of flowering is directly related to fitness and many factors influence it including temperature, photoperiod, resource level, physical stress, and pathogenic infection. The expression of “flowering genes” is regulated by these factors in order to mediate this timing. The FT gene is thought to be expressed in response to all of these factors while the NPR1 gene is induced by salicylic acid (SA), a signaling molecule in the stress-induced flowering pathway. It is unknown whether stress-induced expression of NPR1 may alter expression of specific “flowering genes” such as FT or whether SA may directly influence the expression of FT. Thus, we examined the expression of FT and NPR1 in Arabidopsis thaliana in response to exogenous application of SA. Gene expression was measured using RT-qPCR. Student t-tests were used to assess differences in expression of both FT and NPR1 in response to SA application. FT expression did not significantly differ in response to SA; however, this is likely a result of mispriming due to homology of FT-related genes. Surprisingly, NPR1 expression was not induced by SA in our study. Although our results provide some insights, creative primer design to measure FT expression and modifications to the experimental design are needed to fully elucidate the relationship between FT and NPR1 expression and SA.

CCAAT/enhancer-binding protein- β (C/EBP β ) is a mediator of cell survival and tumorigenesis. When C/EBP β −/− mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19 Arf and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19 Arf is dramatically elevated in C/EBP β −/− epidermis and that C/EBP β represses a p19 Arf promoter reporter. To determine whether p19 Arf is responsible for the proapoptotic phenotype in C/EBP β −/− mice, C/EBP β −/− ;p19 Arf−/− mice were generated. C/EBP β −/− ;p19 Arf−/− mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19 Arf is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBP β −/− epidermis, we generated K14-ER:Ras;C/EBP β −/− mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBP β −/− mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBP β represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBP β may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents.

CCAAT/enhancer-binding protein-beta (C/EBP beta) is a mediator of cell survival and tumorigenesis. When C/EBP beta(-/-) mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19(Arf) and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19(Arf) is dramatically elevated in C/EBP beta(-/-) epidermis and that C/EBP beta represses a p19(Arf) promoter reporter. To determine whether p19(Arf) is responsible for the proapoptotic phenotype in C/EBP beta(-/-) mice, C/EBP beta(-/-); p19(Arf-/-) mice were generated. C/EBP beta(-/-); p19(Arf-/-) mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19(Arf) is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBP beta(-/-) epidermis, we generated K14-ER: Ras; C/EBP beta(-/-) mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBP beta(-/-) mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBP beta represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBP beta may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents.

Recent studies have identified roles for C/EBPβ in cellular survival and tumorigenesis, however, the mechanisms through which C/EBPβ regulates these processes are not fully understood. Previously, we demonstrated that C/EBPβ−/− mice are resistant to carcinogen-induced skin tumorigenesis and in response to topical carcinogen treatment display a 17-fold increase in keratinocyte apoptosis compared to wild-type mice. Here, we have investigated the mechanisms through which C/EBPβ regulates apoptosis in response to carcinogenic stress. Analysis of carcinogen-treated C/EBPβ−/− mouse skin revealed a striking increase in the number of p53 immunopositive keratinocytes in the epidermis of C/EBPβ−/− mice compared to wild-type mice and this increase was temporally associated with a concomitant anomalous increase in apoptosis. The increased levels of p53 were functional as Mdm2, Bcl-2, C/EBPα and p21 were differentially regulated in the epidermis of carcinogen-treated C/EBPβ−/− mice. The increase in p53 protein was not associated with an increase in p53 mRNA levels. To determine whether p53 is required for the increased apoptosis in C/EBPβ−/− mice, C/EBPβ/p53 compound knockout mice were generated. Carcinogen-treated C/EBPβ/p53 compound knockout mice did not display increased apoptosis demonstrating p53 is required for the proapoptotic phenotype in C/EBPβ−/− mice. Our results demonstrate that altered keratinocyte survival in C/EBPβ−/− mice results from aberrant regulation of p53 protein and function and indicate C/EBPβ has a role in the negative regulation of p53 protein levels in response to carcinogen-induced stress.

The basic leucine zipper transcription factor, CCAAT/enhancer binding protein α (C/EBPα) is involved in mitotic growth arrest and has been implicated as a human tumor suppressor in acute myeloid leukemia. We have previously shown that C/EBPα is abundantly expressed in mouse epidermal keratinocytes. In the current study, the expression of C/EBPα was evaluated in seven mouse skin squamous cell carcinoma (SCC) cell lines that contain oncogenic Ha-Ras. C/EBPα mRNA and protein levels were greatly diminished in all seven SCC cell lines compared with normal primary keratinocytes, whereas C/EBPβ levels were not dramatically changed. Reexpression of C/EBPα in these SCC cell lines resulted in the inhibition in SCC cell proliferation. To determine whether the decrease in C/EBPα expression observed in the SCC cell lines also occurred in the carcinoma itself, immunohistochemical staining for C/EBPα in mouse skin SCCs was conducted. All 14 SCCs evaluated displayed negligible C/EBPα protein expression and normal C/EBPβ levels compared with the epidermis and all 14 carcinomas contained mutant Ras. To determine whether oncogenic Ras is involved in the down-regulation of C/EBPα, BALB/MK2 keratinocytes were infected with a retrovirus containing Ras12V, and C/EBPα protein, mRNA and DNA binding levels were determined. Keratinocytes infected with the retrovirus containing oncogenic Ras12V displayed greatly diminished C/EBPα protein, mRNA and DNA binding levels. In addition, BALB/MK2 cells containing endogenous mutant Ras displayed diminished C/EBPα expression and the ectopic expression of a dominant-negative RasN17 partially restored C/EBPα levels in these cells. These results indicate that oncogenic Ras negatively regulates C/EBPα expression and the loss of C/EBPα expression may contribute to the development of skin SCCs.