Mutant-enriched PCR and allele-specific hybridization reaction to detect K-ras mutations in stool DNA: high prevalence in a large sample of older adults.

Background: Testing for mutant K-ras in stool has been proposed for the detection of pancreatic and colorectal cancer (CRC). Different analytical techniques have been developed, but studies of this biomarker in the general population are lacking. We investigated the prevalence and potential determinants of mutant K-ras in stool in a large sample of unselected older adults and assessed the association with colonoscopic findings.

A quantitative assay for aggrecanase activity.

Aggrecanase activities of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases were measured with a recombinant aggrecan fragment and two monoclonal antibodies. Recombinant human aggrecan interglobular domain was first incubated in the presence of ADAMTS enzymes. The aggrecan peptide with the N-terminal sequence ARGSVIL released upon hydrolysis was then quantified in an enzyme-linked immunosorbent assay (ELISA) with an anti-neoepitope antibody specific for the N-terminal ARGSVIL sequence and a second anti-aggrecan peptide antibody.

Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers.

In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the same specimen to assess gene expression during apoptosis.

K-ras mutations in stools and tissue samples from patients with malignant and nonmalignant pancreatic diseases.

Mutant-enriched PCR and reverse dot blot hybridization in microplates were applied for examining K-ras status in stools and tissue samples from patients with pancreatic tumors and chronic pancreatitis. In tissue samples, K-ras mutations were found in 32 of 35 cases of ductal adenocarcinoma, in 5 of 7 periampullary cancers, in 1 cystadenocarcinoma, and in 3 of 5 patients with chronic pancreatitis. In stools, mutated K-ras was seen in 10 of 25 cases of ductal adenocarcinoma, in 1 case of cystadenocarcinoma, and in 2 of 6 cases of chronic pancreatitis.

Fast isolation of RNA to detect expression of tumor markers.

The expression status of several tumor-related proteins is of great interest in clinical examination and research. As a completion to conventional antibody staining, RT-PCR is often used today. Reliable isolation of RNA from a low number of cells is very often a critical stage of such an examination.