Biomaterial Database

Quantitative reverse transcriptase-polymerase chain reaction for prostate-specific antigen mRNA. 1997

B Galvan, and T K Christopoulos

Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.

OBJECTIVE To develop a quantitative reverse transcriptase-polymerase chain reaction assay for monitoring the prostate-specific antigen (PSA) mRNA. METHODS PSA mRNA is amplified, in parallel, with the mRNA of beta-actin, a housekeeping gene. The ratio of the amplification products obtained reflects the relative amount of PSA mRNA with respect to actin mRNA. During PCR, digoxigenin-dUTP is incorporated in the amplified sequences. The PCR products are analyzed separately by time-resolved immunofluorometric hybridization assays, using specific probes immobilized in microtiter wells. The hybrids are reacted with alkaline phosphatase-labeled anti-digoxigenin antibody. The phosphate ester of fluorosalicylate is used as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb(3+)-EDTA which is measured by time-resolved fluorometry. RESULTS The hybridization assays for both PSA and actin amplification products show linearity in the range of 1.4-110 pmol/L. The exponential phase of PCR amplification extends up to 200,000 and 100,000 PSA and actin cDNA molecules, respectively. We prepared mixtures containing various numbers of LNCaP cells in one million cells that do not express PSA and used them as samples in the proposed assay. The ratio of the fluorescence values obtained after analysis of PSA and actin amplification products is linearly related to the number of LNCaP cells in the range of 20 to 3000 cells. Reproducibility studies demonstrate %CVs for the fluorescence ratios of 14.7, 11.8, and 12.2 when samples containing 150, 300 and 1600 LNCaP cells were analyzed (n = 4). CONCLUSIONS A quantitative analytical methodology is provided for monitoring PSA mRNA. The assay is expected to be beneficial in the study of prostate cancer spread.

UI	MeSH Term	Description	Entries
D008297	Male		Males
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012333	RNA, Messenger	RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.	Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D012680	Sensitivity and Specificity	Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed)	Specificity,Sensitivity,Specificity and Sensitivity
D016133	Polymerase Chain Reaction	In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.	Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D017430	Prostate-Specific Antigen	A glycoprotein that is a kallikrein-like serine proteinase and an esterase, produced by epithelial cells of both normal and malignant prostate tissue. It is an important marker for the diagnosis of prostate cancer.	Kallikrein hK3,gamma-Seminoprotein,hK3 Kallikrein,Prostate Specific Antigen,Semenogelase,Seminin,Kallikrein, hK3,gamma Seminoprotein