Preoperative peripheral blood reverse transcription-PCR (RT-PCR) for prostate-specific antigen (PSA) [RT-PCR-PSA] is not associated with an increased risk of progression after radical prostatectomy. We tested the hypothesis that early postoperative peripheral blood RT-PCR-PSA would detect prostate cancer cells persisting in the circulation that would be associated with disease progression. The ...

PCR is widely used for the amplification of DNA and reverse-transcribed RNA. In recent years, real-time PCR has been introduced as a new technique for mRNA quantitation. However, because of the high cost of real-time PCR equipment and supplies, especially using custom-made probes, quantitative RT-PCR is a valuable alternative method. Several quantitative PCR protocols describe the use of a sing...

The development of microfluidic tools for high-throughput nucleic acid analysis has become a burgeoning area of research in the post-genome era. Here, we have developed a microfluidic chip to perform 72 parallel 450-pL RT-PCRs. We took advantage of Taqman hydrolysis probe chemistry to detect RNA templates as low as 34 copies. The device and method presented here may enable highly parallel singl...

Reverse transcriptase polymerase chain reaction (RT-PCR) has become a well-established and powerful molecular technique for studying ribonucleic acids. It is used in medical diagnostics for detecting viral RNA, in hematology and oncology for detecting chimeric transcripts of rearranged genes (1), and in the broad area of research applications in gene expression studies. Since its introduction (...

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In this paper we determine the limits and accuracy of quantitative reverse transcription (RT)-PCR using a modification of the original protocol. The quantification of mRNA with this procedure requires a preliminary estimation of the target molecule (TM) concentration, established from experiments with an internal control molecule (ICM). A definitive quantification is then attained from serial d...

PURPOSE The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated. EXPERIMENTAL DESIGN The "RNA" extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done. Confirmatory RT-PCR experiments used conventionally designed PCR prim...

INTRODUCTION In this protocol, sample and competitor RNAs (previously synthesized as described in Preparation of Competitor RNA for Competitive RT-PCR) are reverse transcribed (separately) in a pilot experiment. A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. This procedure provides an approximate copy number for the sample, ...

The present studies demonstrate a theoretical and practical framework for the accurate quantitation of gene expression in RNA extracted from microscopic tissue samples. The approaches are developed around competitive RT-PCR techniques. Assay performance has been examined and validated at both the RT and PCR steps. Our analysis of RT transcription efficiency for a number of native and competitor...