Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

Background: Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge.

Background: Diagnosis and prognosis in breast cancer are mainly based on histology and immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) material. Recently, gene expression analysis was shown to elucidate the biological variance between tumors and molecular markers were identified that led to new classification systems that provided better prognostic and predictive parameters. Archived FFPE samples represent an ideal source of tissue for translational research, as millions of tissue blocks exist from routine diagnostics and from clinical studies. These should be exploited to provide clinicians with more accurate prognostic and predictive information. Unfortunately, RNA derived from FFPE material is partially degraded and chemically modified and reliable gene expression measurement has only become successful after implementing novel and optimized procedures for RNA isolation, demodification and detection.

The ability to produce diacetyl from pyruvate and L-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from L-and D-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and ␣-aminobutyrate. Pediococcus pentosaceus and Pediococcus acidilactici are homofermentative Gram-positive cocci that produce D-and L-lactate from carbohydrates. These bacteria are regularly found in the nonstarter population of raw milk cheeses at the end of the ripening process. Thus, Pediococcus spp. were isolated from cheddar, ewes' milk cheeses, Comté, AOC Salers, Puzzone di Moena, and various raw milk cheeses traditionally produced in Switzerland, such as Gruyère, Emmentaler, Appenzeller, and Tilsit (1-8).Although several reports have indicated that pediococci alone or combined with lactobacilli can accelerate cheese ripening and enhance flavor development, respectively (9-13), knowledge about metabolic activities affecting the maturation of cheese is limited. Studies on the physiological and biochemical activities of Pediococcus spp. have mainly focused on carbohydrate metabolism and proteolytic and lipolytic activities (14-16). Researchers have also reported that pediococci racemize L-lactate to D-lactate under anaerobic conditions and that pediococci can oxidize lactate to acetate and carbon dioxide under aerobic conditions (17). However, information on the enzymatic degradation of amino acids, important precursors for flavor compounds, is sparse.Diacetyl contributes to desirable flavor in fermented dairy products. A pathway well-known for producing diacetyl is through degradation of citrate (18). In this pathway, pyruvate is formed as an intermediary compound that is then converted via ␣-acetolactate to diacetyl. Although pediococci do not appear to utilize citrate, these species have been reported to form diacetyl (19,20). Pyruvate, which is derived from the metabolism of carbohydrates or amino acids, is likely used for synthesizing diacetyl.In this study, the ability of various strains of P. acidilactici and of P. pentosaceus isolated from cheese to produce diacetyl from pyruvate and serine wa...