Biomedical subjects

Jussi Nurmi

Publications and source records attributed to Jussi Nurmi.

7 recordsLinked to original sources

PubMed2006

Novel homogenous time-resolved fluorometric RT-PCR assays for quantification of PSA and hK2 mRNAs in blood.

OBJECTIVES: The purpose of this study was to design, validate, and optimize internally standardized real-time quantitative RT-PCR assays and to identify and avoid problems with assay reliability and examine the impact of an exogenous internal standard. DESIGN AND METHODS: The model system consisted of internally standardized quantitative real-time RT-PCR assays specific for PSA and hK2 mRNA based on time-resolved fluorometric detection of lanthanide chelates. RESULTS: Reproducibility was best when large copy numbers (>5000 per milliliter blood) were analyzed. Addition of an exogenous target-mimicking internal standard had no significant effect on the reproducibility of the method, but increased the calculated copy numbers by an average of 2-fold. CONCLUSIONS: We developed an internally standardized, specific and reproducible real-time RT-PCR analysis method for PSA and hK2 mRNA in circulating cells in the bloodstream. Both PSA and hK2 assays are sufficiently sensitive to detect two LNCaP cells per milliliter whole blood.

False Positive Reactions↗

PubMed2005

Locked nucleic acid (LNA) probes in high-throughput genetic analysis: application to an assay for type 1 diabetes-related HLA-DQB1 alleles.

OBJECTIVES: In large-scale genetic screening, an assay that is reliable, fast and easy to perform, and straightforwardly adapted to new analytes is a necessity. We describe a one-step assay for analyzing HLA-DQB1 alleles which are associated with susceptibility to type 1 diabetes. DESIGN AND METHODS: The assay is based on asymmetric PCR amplification and a homogeneous hybridization method. The specificity of the probes was improved by substituting LNA (locked nucleic acid) for DNA at the critical bases. RESULTS: The functionality of the LNA containing probes was found to be superior compared to probes consisting of DNA only. The homogeneous assay gave a correct genotyping result in 100% of the cases, which included both extracted DNA samples and blood samples dried on sample collection cards. CONCLUSION: This homogeneous approach provides a simple method to define disease risk associated with HLA alleles for large-scale screening projects.

Base Sequence↗

PubMed2005

The genomics of probiotic intestinal microorganisms.

An intestinal population of beneficial commensal microorganisms helps maintain human health, and some of these bacteria have been found to significantly reduce the risk of gut-associated disease and to alleviate disease symptoms. The genomic characterization of probiotic bacteria and other commensal intestinal bacteria that is now under way will help to deepen our understanding of their beneficial effects.

Bacteria↗

PubMed2005

In vitro effects on polydextrose by colonic bacteria and caco-2 cell cyclooxygenase gene expression.

A 4-stage colon simulator and a cell culture-based human intestinal epithelial function model were combined to study the effects of a soluble fiber, polydextrose (PDX), on intestinal microbes and mucosal functions relevant to the risk of colon cancer. We observed sustained degradation of PDX throughout the different stages of the model. The fermentation was characterized by gradual degradation of PDX, production of short-chain fatty acids, and no increasing in putrefactive markers. We observed less marked effects in the microbial densities. When we applied colon fermentation metabolites obtained from the simulators with PDX to Caco-2 colon cancer cell line, a significant dose-dependent decreasing effect on cyclooxygenase-2 (COX-2) and an increasing effect on COX-3 expression levels were observed. PDX concentration appeared not to have effect on the expression levels of COX-1. Overexpression of COX-2 and decreased expression of COX-1 have been suggested to be characteristics of colon cancer. The exact physiological role of COX-3, an intron-retaining splice variant of COX-1, is not known, but it is suspected to play a role in transcriptional regulation of COX-1 and COX-2. In vitro modulation of COX expression by colon microbial fermentation products of polydextrose offers an interesting starting point for further studies on possible risk-decreasing effect of PDX on the development of colon cancer.

Bacteria↗

PubMed2003

A homogeneous high-throughput genotyping method based on competitive hybridization.

OBJECTIVES: A reliable high-throughput assay system is necessary for the analysis of the ever-increasing numbers of single-nucleotide polymorphisms (SNP) relevant to genetic screening studies. We describe an assay suitable also for large-scale screening programs. DESIGN AND METHODS: The one-step assay is based on asymmetric PCR amplification of the target sequence and subsequent time-resolved fluorescence measurement. Asymmetric amplification results in a single-stranded PCR product that is detected in the amplification vessel with a highly sensitive, homogeneous hybridization method. RESULTS: A dual label, homogeneous high-throughput platform for nucleic acid sequence analysis was developed and validated using a C/T single-nucleotide polymorphism in the insulin gene as a model analyte and applied also to two other SNP-assays (poliovirus receptor A/G-polymorphism and CD86-gene exon 2 A/G-polymorphism). CONCLUSIONS: The described high-throughput genotyping technology is very competitive in price, simple in design and easily applied to any analyte sequence.

Genetic Testing↗

PubMed2002

High-performance real-time quantitative RT-PCR using lanthanide probes and a dual-temperature hybridization assay.

We report a time-resolved fluorescence-based, homogeneous approach for multiplex, real-time or end-point detection of PCR products. Signal generation consists of PCR associated digestion of a 5'-labeled oligonucleotide probe, rapid cooling of the reaction mixture, and hybridization of undigested probe oligonucleotides with a complementary, shorter probe that incorporates a quencher at its 3' end. The signal coming from intact fluorescent probe molecules is, thus, quenched. The fluorophores we have used are environmentally sensitive lanthanide chelates. Their signals can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. Signal-to-noise ratios are further enhanced by the environmental sensitivity of these chelates; they exhibit a higher fluorescence intensity when free in solution than when coupled to intact probe molecules. Because of the minimal background fluorescence, the signal-to-noise ratios are higher and threshold cycles are lower than those obtained using conventional TaqMan probes. The multiplexing capacity of the assay chemistry is demonstrated through simultaneous amplification and detection of prostate specific antigen (PSA) cDNA and an internal standard mRNA (mmPSA) using probes labeled with terbium and europium. The applicability of the assay chemistry to routine clinical diagnostics is demonstrated through absolute quantification of PSA mRNA in peripheral blood.

Europium↗

PubMed2002

Simultaneous quantification of prostate-specific antigen and human glandular kallikrein 2 mRNA in blood samples from patients with prostate cancer and benign disease.

BACKGROUND: Detection or quantification of circulating cancer cells has been proposed as an aid in detection and monitoring of several solid tumors. We investigated the classification accuracy of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) mRNA copy numbers in blood for the differentiation of patients with prostate cancer (PC) and benign disease. METHODS: PSA and hK2 mRNA expression was studied in blood samples from 51 men with PC and 19 men with benign disease. Among the PC patients, 10 had organ-confined disease (pT1-pT2). We used a multiplexed reverse transcription-PCR assay with two highly target-like mRNA internal standards for the simultaneous quantification of PSA and hK2 mRNA. An external calibration curve covered the range of 10(2)-10(6) mRNA copies. RESULTS: PSA and hK2 mRNA were detected in 41 of 51 (median, 1200 copies/0.5 mL of blood) and 43 of 51 (median, 3800 copies/0.5 mL of blood) patients with PC, respectively, whereas only 1 of 19 men with benign disease was positive for both mRNAs (1500 PSA and 3100 hK2 mRNA copies/0.5 mL of blood; P <0.0001, Mann-Whitney U-test). Of the 10 patients with organ-confined PC, only 3 with low Gleason scores (< or =5) were negative for both PSA and hK2 (P = 0.02, Mann-Whitney U-test). CONCLUSIONS: The presence of PC cells in the blood circulation is an early event in PC progression, and quantitative assays for PSA and hK2 mRNA discriminate benign from PC cases. Further studies are needed to determine the diagnostic accuracy and prognostic value of the assays.

Aged↗