A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of <0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.Polymerase chain reaction (PCR) is a powerful tool to amplify small amounts of DNA or mRNA for various molecular analyses. It has been used for RNA blot analysis (1), nuclease protection analysis (2), and mRNA phenotyping (3) for the study of short-lived, low-copy number, mRNA transcripts. It has widespread applications in genetic disease diagnosis (4, 5), disease susceptibility (6, 7), and cancer diagnosis (1,8). However, in most instances, the PCR technique has only provided qualitative results. The availability of quantitative PCR should provide valuable additional information for these and other applications.Quantitative PCR analyses have been used to study the mRNA levels for dystrophin in different tissues (9) and for thymidylate synthase in tumors (10). However, these studies provide only relative comparisons of the amounts of mRNA. It has been difficult to quantitate the absolute amount of specific mRNA without an internal standard of known concentration. Because PCR amplification is an exponential process, the extent of amplification (N) is given by the equation N = NO(l + eff)y, where No is the initial amount of material, eff is the efficiency, and n is the cycle number. Small differences in efficiency could lead to large differences in the yield of PCR product. Previous attempts to quantitate PCR amplification of mRNA sequences have involved the use of a relatively invariant mRNA such as 8-actin or an unrelated template as an internal standard (9, 11). However, this approach provides only comparative data, in part because ofdifferences in efficiency between the primer pairs for the standard and the target mRNAs. Ideally, target mRNA could be quantified most accurately by using an internal standard with the same sequence as the target itself. However, to control for "tube effects," the standard and the target RNAs must be amplified in the same reaction tube. Unfortunately, in this case, their PCR products cannot be distinguished. A second approach is to generate an allelic variant (e.g., a small deletion or insertion in the gene of interest) such that there is a small difference in the size of the PCR product of this interna...

Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.

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The prediction of tumour biology rarely rests upon a single characteristic of the malignancy. The analysis of a single gene can complement standard histologic evaluation. The investigation of new parameters as well as the routine clinical analysis of gene expression is often limited because of the small amount of tissue available. This is particularly true of de novo human bladder cancers because they are generally small or handled in such a way as to hinder the analysis of multiple different parameters. Analysis of expressed mRNA by the polymerase chain reaction (RNA/PCR) is a method that allows the development of a profile of bladder cancer gene expression. The authors report the use of the RNA/PCR method to examine in bladder cancer the expression of the human leukocyte antigen (HLA) class II gene family (HLA-DR, DQ, and DP) as well as interleukin-6 (IL-6) and the interleukin-6 receptor (IL-6R). All de novo transitional cell carcinomas, one squamous carcinoma, and two transitional cell carcinoma cell lines expressed the majority of HLA class II genes. All samples expressed IL-6R RNA whereas production of IL-6 message was limited to one of the cell lines and to the high-grade bladder cancers. These results were combined with stage, grade, and DNA content to develop a profile of the cancers examined. Although an improved predictive index based on gene expression analysis by RNA/PCR has not been realized, a broader survey of human tumors for expression of these genes and others is likely to refine the classification of bladder cancer.

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