Abstract
A quantitative polymerase chain reaction (PCR) method for determining concentrations of mRNA for the cyclic AMP (cAMP)-binding protein RI alpha, a regulatory subunit of cAMP-dependent protein kinase, was developed using site-directed mutagenic primers and mix-melt PCR. The PCR product for RI alpha mRNA was modified to include an EcoRV restriction site for use as an internal standard. This mutant utilised the same primers as the target mRNA and differed in sequence by only four bases. As only one of these base changes results in a purine/pyrimidine switch the effective change in labelling with [32P]dCTP was less than 0.5%. Reverse transcription of mRNA was performed and quantitative PCR was carried out using fixed levels of mutant RI alpha vs varying amounts of both normal RI alpha sequence of known concentration and unknown samples. Validation of the technique using rigourous quality control established that reverse transcription, determined by incorporation of labelled nucleotides, gave intra- and interassay variations of 16.2 and 9.3% respectively. Using crossover evaluation of cDNA concentrations with cloned RI alpha sequences as controls intra- and interassay variations of 14.3% and 4-8% respectively were obtained. Using compounded errors, the limits of precision for this technique demonstrate that values that are altered by 50% or more represent a true alteration in mRNA levels between samples tested. This value compares favourably to similar values for radioimmunoassays of between 10% and 30% precision. Analysis of a series of patient samples during routine follow-up of treatment demonstrated clear changes in mRNA levels. Using site-directed mutagenesis to establish a quantitative PCR-based assay for expression of mRNA this study demonstrates the potential usefulness and some limitations of quantitative PCR for applications within a clinical biochemistry laboratory. However, based on compounded error, values that vary by less than 50% within assays, and by less than 70% in separate assays could not be clearly separated. Assessment of paired patient samples has demonstrated clear changes in mRNA for the target protein RI alpha. With the use of normal quality control procedures this study has established that the degree of reproducibility of this quantitative PCR technique would allow assessment of mRNA levels for markers of interest in clinical samples in a routine laboratory context.
Highlights
'UniversitY Department of Surgery, Glasgows RoYal Infirmarn, Glasgows G31 2ER; -ICRF Medical Oncology U-nit, Western General
Reverse transcription of mRNA was performed and quantitatisve polvmerase chain reaction (PCR) was carried out using fixed levels of mutant RI alpha *s varying amounts of both normal RI alpha sequence of know-n concentration and unknown samples
Using compounded errors. the limits of precision for this technique demonstrate that values that are altered by 500o or more represent a true alteration in mRNA levels betw-een samples tested
Summary
The sequence for the cDNA of human cAMP-dependent protein kinase type-I-alpha subunit isolated from human testis (Sandberg et al.. 1990) wxas retrieved from the Gembl database (accession no: M33336). (1) Five RNA samples u-ere reverse transcribed as described above. PCR reactions u-ere performed in a final volume of 100 p1 containing the following: 0.5 units of Taq poly'merase Flanking the central portion of the cAMP RI alpha-binding protein PCR amplimer. These oligonucleotides contained an EcoRV restriction site to be introduced by mismatch PCR into the amplimer. The full mutation containing 430 bp RI alpha amplimer was synthesised follo'ing melting and reannealing of the 3' and 5' portions followed by PCR of the annealed DNA using the normal flanking primers for the RI alpha 430 bp fragment (Figure 1)
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