A NOVEL, RAPID IN CELL RNA AMPLIFICATION TECHNIQUE FOR THE DETECTION OF LOW COPY MESSENGER-RNA TRANSCRIPTS

Growing interest now focuses on improvements of in situ polymerase cha in reaction (PCR) technology for the detection of DNA and RNA cellular sequences. In this study, reverse transcription PCR in situ hybridisa tion (RT PCR-ISH) was developed and used to determine gene expression of pyruvate dehydrogenase in a cell model system, using human peripher al blood lymphocytes (PBLs). The success of in cell RNA amplification depends on the type of cell/ tissue fixation, cell permeabilisation, a nd the efficiency of reverse transcription and cDNA amplification. Thi s paper presents new approaches to overcome the critical aspects of fi xation, permeabilisation, and reverse transcription when performing in cell RNA amplification. A novel fixative, ''Permeafix'', possessing f ixative and permeabilisation properties, was used for cell fixation pr ocedures. ''Permeafix'' obviated the need for pre-amplification proteo lysis, facilitating entry of PCR reagents to target sequences within t he cell. In addition, a simple one step RNA in cell amplification prot ocol using recombinant Thermus thermophilus (rTth) DNA polymerase, whi ch reverse transcribes mRNA efficiently to cDNA and then catalyses cDN A amplification, was used, The value of a semi-junctional primer syste m for in cell gene expression studies, without the need to perform DNa se digestion, is demonstrated.