Re-amplification of differentially expressed mRNA fragments of head-neck cancers without cloning

Abstract

mRNA expression of healthy and malignant cells can be compared to each other by employing the "differential display" (DD) technique. Most studies describe sequence analysis of differentially expressed fragments after reamplification by a second round of PCR and subsequent molecular cloning to gain a sufficient amount of DNA for sequencing. The aim of this study was to show whether a sufficient amount of differentially expressed mRNA of squamous cell carcinoma cells of the head and neck region can be generated by PCR alone without cloning steps. mRNA isolated from cultivated keratinocytes and squamous cell carcinoma cells was reverse transcribed into cDNA which was amplified with PCR. Differentially expressed fragments detected after gel electrophoresis were isolated from the gel and reamplified in a second PCR. The resulting cDNA amounts of the second PCR were suitable for cloning but not for direct sequencing. A third round of PCR with the undiluted final product of the second PCR as template regularly failed. Dilutions of the second PCR products between 1:10 and 1:10(10) were prepared. The third round of PCR was carried out with these various template concentrations. A sufficient amount of differentially expressed fragments for sequencing procedures resulted when dilutions of the second PCR products ranging from 1:10(2) to 1:10(7) were used as templates in the third round of PCR. Modifications of PCR parameters provide high DNA copy numbers of differentially expressed mRNA fragments from squamous cell carcinoma cells of the upper aerodigestive tract in amounts that are needed for sequence analysis. This may make it possible to avoid labor-intensive cloning procedures requiring high safety standards.