Biomedical subjects

Michael W Pfaffl

Publications and source records attributed to Michael W Pfaffl.

18 recordsLinked to original sources

PubMed2006

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR.

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.

Algorithms↗

PubMed2006

Distribution-insensitive cluster analysis in SAS on real-time PCR gene expression data of steadily expressed genes.

Cluster analysis is a tool often employed in the micro-array techniques but used less in the real-time PCR. Herein we present core SAS code that instead of the Euclidian distances takes correlation coefficient as a dissimilarity measure. The dissimilarity measure is made robust using a rank-order correlation coefficient rather than a parametric one. There is no need for an overall probability adjustment like in scoring methods based on repeated pair-wise comparisons. The rank-order correlation matrix gives a good base for the clustering procedure of gene expression data obtained by real-time RT-PCR as it disregards the different expression levels. Associated with each cluster is a linear combination of the variables in the cluster, which is the first principal component. Large set of variables can then be replaced by the set of cluster components with little loss of information. In this way, distinct clusters containing unregulated housekeeping genes along with other steadily expressed genes can be disclosed and utilized for standardization purposes. Simulated data in parallel with the data from a biological experiment were taken to validate the SAS macro. For both cases, good intuitive results were obtained.

Cluster Analysis↗

PubMed2006

RNA integrity and the effect on the real-time qRT-PCR performance.

The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Working with low-quality RNA may strongly compromise the experimental results of downstream applications which are often labour-intensive, time-consuming, and highly expensive. Using intact RNA is a key element for the successful application of modern molecular biological methods, like qRT-PCR or micro-array analysis. To verify RNA quality nowadays commercially available automated capillary-electrophoresis systems are available which are on the way to become the standard in RNA quality assessment. Profiles generated yield information on RNA concentration, allow a visual inspection of RNA integrity, and generate approximated ratios between the mass of ribosomal sub-units. In this review, the importance of RNA quality for the qRT-PCR was analyzed by determining the RNA quality of different bovine tissues and cell culture. Independent analysis systems are described and compared (OD measurement, NanoDrop, Bioanalyzer 2100 and Experion). Advantage and disadvantages of RNA quantity and quality assessment are shown in performed applications of various tissues and cell cultures. Further the comparison and correlation between the total RNA integrity on PCR performance as well as on PCR efficiency is described. On the basis of the derived results we can argue that qRT-PCR performance is affected by the RNA integrity and PCR efficiency in general is not affected by the RNA integrity. We can recommend a RIN higher than five as good total RNA quality and higher than eight as perfect total RNA for downstream application.

Animals↗

PubMed2005

Effects of varied EGCG and (+)-catechin concentrations on proinflammatory cytokines mrna expression in cona-stimulated primary white blood cell cultures.

EGCG [(-)-epigallocatechin gallate] and (+)-catechin hydrate are flavanoids, which are known as anticancer and healthy drugs. To test the immune modulatory effects of EGCG and catechin, various concentrations were tested on primary white blood cells (WBC) in cell cultures stimulated with the T-cell mitogen concanavalin A (ConA). WBC from dairy cows (1 x 10(6) cells/mL) were cultivated using RPMI medium with FCS and gentamycin. First, WBC were stimulated with ConA, and 6 h later the flavanoid treatment was started. Cultivated WBC were treated with various physiological flavanol concentrations (0-100 microM) in cross-combination with various ConA concentrations (0-1 microg/mL). After 24 h, cells were harvested, cell viability was verified, and total RNA was isolated. Relative mRNA expression levels of proinflammatory cytokines TNFalpha, IL1beta, IL6, and transcription factor cFos and of nucleosome component histon H3 were quantified with real-time qRT-PCR. High EGCG and catechin concentrations had inhibitory effects on total RNA expression. Low EGCG concentration can induce total RNA expression in WBC. EGCG reduced cFos mRNA expression, which can be abolished by high ConA concentrations in a reverse dose-dependent manner. TNFalpha showed a flavanoid-specific expression pattern. EGCG acts in blood physiological concentrations (micromolar range), and catechin acts in higher gut-relevant concentrations (millimolar range) and has the potential to influence the proinflammatory TNFalpha expression. Higher flavanoid concentration had more pronounced effects than lower, whereas EGCG showed a more potent suppression of gene expression than catechin (toward TNFalpha). EGCG and catechin had no significant effects in primary WBC on the expression pattern of the proinflammatory cytokines IL1beta and IL6 and on the expression of the housekeeping genes GAPDH and histon H3. It is presumed that both flavanoids have the potential to regulate total RNA expression and gene-specific expression in WBC.

Animals↗

PubMed2005

Model of inhibition of Thermus aquaticus polymerase and Moloney murine leukemia virus reverse transcriptase by tea polyphenols (+)-catechin and (-)-epigallocatechin-3-gallate.

Non-nutritional polyphenolic compounds such as (+)-catechin and (-)-epigallocatechin-3-gallate (EGCG) are known as anticancer chemopreventive agents and have been utilised for medical purposes in form of tea drinking. Documented anticancer properties of these compounds result from their antioxidant effects. However, also direct alteration of an enzyme performance has been reported and deserves more attention. In this paper, a direct effect of catechin and EGCG on the performance of reverse transcription (RT) and/or polymerase chain reaction (PCR) was studied. Both tea polyphenolic compounds were added into real-time RT-PCR reactions and the fluorescence data obtained were fitted with a mathematical model. Several parameters of PCR performance were compared, obtained from the mathematical model for reactions with and without addition of (+)-catechin and EGCG. Addition of EGCG to enzyme reaction seems to inhibit the RT reaction (p < 0.05) and to slow down the DNA polymerase reaction (p < 0.001). Similarly, (+)-catechin inhibited the DNA amplification (p < 0.01) but had no effect on the RT reaction. The effects could be observed in physiological flavanol concentrations ranging from 10(-5) to 10(-8)M.

Animals↗

PubMed2005

Effects of increased cellular zinc levels on gene and protein expression in HT-29 cells.

High cellular zinc concentrations lead to impairments in ATP synthesis and cell cycle control particularly in neurons and epithelial cells. The molecular basis for these dysfunctions is still not fully elucidated. Here we analyzed the effects of a high zinc exposure (10 ppm) on gene and protein expression in the human epithelial cell line HT-29. Of the 1176 genes analyzed with cDNA arrays, nine differentially expressed genes were identified. Proteome analysis based on 1310 detected proteins identified 11 molecular targets. Most of the identified genes/proteins have not been linked to cellular zinc status before (e.g. PEC-60, R-ras3). More than half of the targets participate in ATP production or stress response. Therefore, it appears that higher zinc concentrations mediate their effects mainly via impairments in cellular energy metabolism and stress response.

Gene Expression Profiling↗

PubMed2004

Elevated caspase-3 and Fas mRNA expression in jejunum of adult rats during subclinical zinc deficiency.

The programmed cell death-so-called apoptosis-is a physiological process occurring in all multicellular organisms to control cell-number homeostasis. Nevertheless, increase of apoptotic cell death in different organs can lead to pathological alterations. As zinc is a potent inhibitor of apoptosis, we investigated the influence of zinc deficiency on mRNA expression levels of caspase-3 and Fas in adult rats. For this purpose, 24 adult rats fed a Zn-deficient diet for up to 29 days were compared to seven animals in the control group. After 1, 2, 4, 7, 11, 16, 22 and 29 days of treatment three animals were sacrificed (n = 24). Total RNA extraction from thymus, liver, jejunum and colon was carried out. Samples were reverse transcribed and subjected to real-time PCR. Relative quantification of caspase-3 and Fas mRNA expression was achieved on the basis of normalisation by glycerolaldehyd-3-phosphate-dehydrogenase mRNA expression levels in all samples. In jejunum, up to day 11 the relative mRNA expression of the respective genes decreased. A significant increase in caspase-3 and Fas expression was found from day 11 of zinc deficiency onward. In contrast, mRNA expression in liver and colon remained unaffected, whereas thymus showed a slight but not significant increase in the expression of these genes. This study provides the first evidence that even moderate zinc deficiency in an adult, non-growing rat model is able to elevate mRNA expression levels of factors involved in early stages of apoptosis.

Animals↗

PubMed2004

Inhibition of real-time RT-PCR quantification due to tissue-specific contaminants.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) is currently considered the most sensitive method to study low abundance gene expression. Since comparison of gene expression levels in various tissues is often the purpose of an experiment, we studied a tissue-linked effect on nucleic acid amplification. Based on the raw data generated by a LightCycler instrument, we propose a descriptive mathematical model of PCR amplification. This model allowed us to study amplification kinetics of four common housekeeping genes in total RNA samples derived from various bovine tissues. We observed that unknown tissue-specific factors can influence amplification kinetics but this affect can be ameliorated, in part, by appropriate primer selection.

Animals↗

PubMed2004

Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations.

The stability of standard gene expression is an elementary prerequisite for internal standardisation of target gene expression data and many so called housekeeping genes with assumed stable expression can exhibit either up- or down-regulation under some experimental conditions. The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index. The index can be compared with further ten target genes to decide, whether they are differentially expressed under an applied treatment. All data processing is based on crossing points. The BestKeeper software tool was validated on four housekeeping genes and 10 members of the somatotropic axis differentially expressed in bovine corpora lutea total RNA. The BestKeeper application and necessary information about data processing and handling can be downloaded on http://www.wzw.tum.de/gene-quantification/bestkeeper.html.

Animals↗

PubMed2004

Effects of mycophenolic acid (MPA) treatment on expression of Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIgR) mRNA in adult sheep tissues.

AIM: To quantify the expression of Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIgR) mRNA under a long-term influence of mycotoxin mycophenolic acid (MPA), which is used in human transplantation medicine due to its immunosuppressive properties and is a common contaminant in silage. METHOD: We applied 300 mg MPA/day in nine sheep for nine weeks and compared them with untreated animals (n=9). The expression level of Ig receptor mRNA was determined in eight different adult ovine tissues (liver, kidney, jejunum, ileum, spleen, thymus, mesenteric and pharyngeal lymph nodes). For a reliable and sensitive mRNA quantification of Ig receptor subtypes, a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used with a relative- and tissue-specific efficiency corrected quantification model (REST). RESULTS: Each tissue exhibited an individual expression pattern of FcRn and pIgR mRNA. Both types of Ig receptors were highly expressed in the liver, kidney, and gastrointestinal tract. Medium-to-low expressions were found in the spleen, thymus, mesenteric and pharyngeal lymph nodes. FcRn mRNA was significantly down-regulated by MPA in the liver (p=0.02). After MPA treatment, a significant up-regulation of pIgR mRNA expression was observed in the ileum and liver (p=0.04 for both). Expression level of FcRn mRNA in the tissues in decreasing order was as follows: liver > kidney > jejunum > ileum > spleen > thymus > mesenterial lymph node > pharyngeal lymph node; whereas expression level for pIgR mRNA was the following: liver > kidney > jejunum > ileum > pharyngeal lymph node > spleen > thymus > mesenterial lymph node. CONCLUSION: The MPA exhibited immunomodulatory effects in the liver and ileum of treated sheep. Its possible immunosuppressive effects may be explained by lowering the level of FcRn expression in the liver, which resulted in a lower IgG serum-to-bile transport. However, MPA showed stimulatory effects on pIgR expression in the liver and ileum, suggesting good IgA and IgM transport in these tissues.

Animals↗

PubMed2003

Standardized determination of real-time PCR efficiency from a single reaction set-up.

We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.

Algorithms↗

PubMed2003

Effect of zinc deficiency on the mRNA expression pattern in liver and jejunum of adult rats: monitoring gene expression using cDNA microarrays combined with real-time RT-PCR.

In the study presented here, the effect of zinc deficiency on mRNA expression levels in liver and jejunum of adult rats was analyzed. Feed intake was restricted to 8 g/day. The semi-synthetic diet was fortified with pure phytate and contained either 2 microg Zn/g (Zn deficiency, n = 6) or 58 microg Zn/g (control, n = 7). After 29 days of Zn depletion feeding, entire jejunum and liver were retrieved and total RNA was extracted. Tissue specific expression pattern were screened and quantified by microarray analysis and verified individually via real-time RT-PCR. A relative quantification was performed with the newly developed Relative Expression Software Tool Copyright on numerous candidate genes which showed a differential expression. This study provides the first comparative view of gene expression regulation and fully quantitative expression analysis of 35 candidate genes in a non-growing Zn deficient adult rat model. The expression results indicate the existence of individual expression pattern in liver and jejunum and their tissue specific regulation under Zn deficiency. In addition, in jejunum a number of B-cell related genes could be demonstrated to be suppressed at Zn deficiency. In liver, metallothionein subtype 1 and 2 (MT-1 and MT-2) genes could be shown to be dramatically repressed and therefore represent putative markers for Zn deficiency. Expression results imply that some genes are expressed constitutively, whereas others are highly regulated in tissues responsible for Zn homeostasis.

Animals↗

PubMed2003

Tissue-specific expression pattern of bovine prion gene: quantification using real-time RT-PCR.

In recent studies PrP mRNA was determined mostly by in situ hybridisation or Northern Blot analysis--methods not suitable for absolute quantification of mRNA copy numbers. Herein we report on bovine prion mRNA quantification using calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different regions of the CNS and seven peripheral organs. PrP(c) mRNA copy numbers could be determined in all tissues under study. In approval with prior studies high mRNA level was found in Neocortex and Cerebellum. Lymphatic organs showed at least as high expression levels of prion mRNA as overall brain. Lowest expression was detected in kidney. Results of our study provide insight into the involvement of different organs in pathogenesis with respect to prion mRNA expression. LightCycler technology is currently considered the most precise method for nucleic acid quantification and showed to be powerful tool for further studies on prion diseases pathogenesis.

Animals↗

PubMed2003

Influence of zinc deficiency on the mRNA expression of zinc transporters in adult rats.

The accumulation of zinc in the cell is a sum of influx and efflux processes via transporter proteins, like the four Zn transporters (ZnT1-4), the divalent cation transporter 1 (DCT1) and of storage processes mainly bound to metallothionein (MT). To study the effect of Zn deficiency on mRNA expression levels, adult rats were used as an animal model. Food intake was restricted to 8 g/day containing 2 microg Zn/g fortified with pure phytate in Zn deficiency rats and 58 microg Zn/g in controls (n = 7). At day 1, 2, 4, 7, 11, 16, 22, and 29 of Zn deficiency, 3 animals were sacrificed, respectively (n = 24). Zn deficiency was evident from reduced plasma Zn, plasma alkaline phosphatase activity and severe mobilization of Zn from tissue stores (mainly skeleton), while food intake and body weight remained unaffected. Tissues representing Zn absorption (jejunum, colon), Zn storage and utilization (muscle, liver), and Zn excretion (kidney) were retrieved. Total RNA contents increased in colon (p = 0.003) and trend to decrease in liver (p = 0.086). Zn deficiency was without effect on tissue total RNA concentrations in muscle tissue and kidney. Real-time reverse transcription (RT) polymerase chain reaction (PCR) assays were developed and a relative quantification on the basis of GAPDH was applied. Assays allowed a relative and accurate quantification of mRNA molecules with a sufficiently high sensitivity and repeatability. All known Zn transporter subtypes were found in the tissues. ZnT3 was newly elucidated and sequenced in rat tissues. Expression patterns and reactions to Zn deficiency were specific for the tissue analysed. Expression results imply that some transporters are expressed constitutively, whereas others are highly regulated in tissues responsible for Zn homeostasis. The most distinct changes of expression levels were shown in colon which can therefore be postulated as a highly Zn sensitive tissue. MT was down-regulated in all tissues, massively in liver (p < 0.001) and in colon (p = 0.002) and in tendency also in the jejunum and kidney. In parallel with intracellular Zn status it is a potent candidate gene for Zn deficiency. ZnT1 and ZnT2 showed a significant up-regulation of mRNA expression in colon (p = 0.032 and p = 0.026) and for ZnT2 a trend of down regulation in jejunum (p = 0.098). This study provides the first comparative view of regulation of gene expression and fully quantitative expression analysis of all known Zn transporters in a non growing adult rat model on a constant platform and therefore allows a direct comparison.

Animals↗

PubMed2003

Non-invasive screening for treatment of heifers with the anabolic steroid melengestrol acetate (MGA) by feces analysis.

For eight weeks, two heifers each had been orally administered daily doses of 0, 1.5, or 5 mg melengestrol acetate (MGA) in a feed premix. Four heifers received the labeled dose of 0.5 mg/day. Regular feces samples were taken throughout the experiment. A rapid screening method for the determination of MGA in feces was developed, consisting of liquid extraction, clean-up on solid-phase extraction cartridges and quantification by enzyme immunoassay (ELISA). Residues in feces were dose-dependent with mean values of < 0.25, 2.0, 4.4, or 15.4 ng/g for 0, 0.5, 1.5, and 5 mg/day doses, respectively. In contrast to urine analysis, feces analysis appeared to be a suitable means of non-invasive screening before slaughter for surveillance of MGA treatment and verification of its compliance with labeled dosage.

Administration, Oral↗

PubMed2003

Quantitative mRNA analysis of eight bovine 5-HT receptor subtypes in brain, abomasum, and intestine by real-time RT-PCR.

Serotoninergic pathways are involved in economically important bovine gastrointestinal (GI) motility disorders such as displaced abomasum and cecal dilatation/dislocation. The existing research tools to investigate the role of serotoninergic pathways in such disorders in ruminants comprise functional pharmacological methods, e.g., in vitro contractility studies in tissue baths, and electromyographical recordings in vivo. However, no tools for quantification of bovine serotonin receptor [5-hydroxytryptamine receptor (5-HTR)] expression were available so far. This study aimed to develop real-time RT-PCR assays for quantitative mRNA analysis of bovine 5-HTR subtypes. Because the bovine 5-HTR coding sequences (CDSs) were completely unknown, multiple species (human, mouse, and rat) alignment of complete CDS was used for primer design in highly homologous regions. LightCycler real-time RT-PCR assays (partial CDS) for the following bovine 5-HTR subtypes were developed and validated: 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1F, 5-HTR2A, 5-HTR2B, 5-HTR2C, and 5-HTR4. Intra- and inter-assay coefficients of variation (CV) for the eight established assays were small, ranging from 0.49% to 2.46%. As a first physiological application, 5-HTR mRNA expression levels were measured in brain, abomasum, and intestine of 10 healthy, lactating dairy cows. The 5-HTR expression was quantified by normalization to the housekeeping gene glyceraldehyde-phosphate-dehydrogenase (GAPDH). The 5-HTR subtype expression levels ranged from 0.001% (5-HTR2C in intestine) to 1% 5-HTR/GAPDH (5-HTR1B and 5-HTR4 in intestine). There were high variations of 5-HTR subtype mRNA expression within tissues across receptor subtypes and within receptor subtypes across tissues. In conclusion, accurate real-time RT-PCR assays for quantitative analysis of bovine 5-HTR subtype gene expression were developed and validated.

Abomasum↗

PubMed2002

Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.

Real-time reverse transcription followed by polymerase chain reaction (RT-PCR) is the most suitable method for the detection and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene. Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the calculation of relative expression ratio allow only for the determination of a single transcription difference between one control and one sample. Therefore a new software tool was established, named REST (relative expression software tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance by a randomisation test. Herein, development and application of REST is explained and the usefulness of relative expression in real-time PCR using REST is discussed. The latest software version of REST and examples for the correct use can be downloaded at http://www.wzw.tum.de/gene-quantification/.

Animals↗

PubMed2002

Expression of estrogen and progesterone receptors in the bovine ovary during estrous cycle and pregnancy.

The objective of the study was to demonstrate the mRNA expression of estrogen receptor a (ERa), ERbeta, and progesterone receptor (PR) by block reverse transcription-polymerase chain reaction(RT-PCR) and real-time RT-PCR (LightCycler) in bovine ovarian follicles and in corpus luteum during the estrous cycle and pregnancy. The mRNA expression of ERalpha and ERbeta mRNA in theca interna tissue (TI) (lower pg/microg RNA) increased continuously and significantly during final growth of follicles, with much higher levels for ERalpha. The mRNA expression of ERalpha and ERbeta in granulosa cells (GC) (fg/microg RNA) increased continuously during follicle growth but without any significant change. The expression of mRNA for PR in follicles (lower fg/microg RNA) increased continuously to maximum level in preovulatory follicles with a significant change only in TI. The highest mRNA expression for ERalpha (fg/microg RNA) was detected in corpus luteum (CL) during the early luteal phase, following by a significant decrease of expression during the mid, late, and regression phases. In contrast, ERbeta mRNA expression is relatively high during the early stage, decreased during the late early and mid luteal phase, and increased significantly again during the late luteal phase and after CL regression. During pregnancy (>3 mo), low levels of ERalpha and ERbeta mRNA expression (<25 fg/microg RNA) with no significant changes were measured. No significant change in PR mRNA expression (levels <13 fg/microg RNA) during the estrous cycle and pregnancy in bovine CL were found. The results suggest an autocrine/paracrine role of steroid receptors in the regulation of final follicle growth and corpus luteum formation and function.

Animals↗