QUANTIFICATION OF VITAMIN-D-RECEPTOR MESSENGER-RNA IN TISSUE-SECTIONSDEMONSTRATES THE RELATIVE LIMITATIONS OF IN-SITU REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION

In situ-reverse transcriptase-polymerase chain reaction (IS-RT-PCR) is a recently described technique that is used to localize low levels of mRNA within cells and tissue sections. One of the major criticisms le velled at this technique is that positive results may be meaningless, as amplification is required to demonstrate the transcripts of interes t. The use of IS-RT-PCR to demonstrate mRNA for receptors for 1,25-dih ydroxyvitamin D-3 (VDR) in sections of human kidney and bone has previ ously been described. To ascertain whether the levels of VDR mRNA dete cted following IS-RT-PCR mere transcriptionally significant, computeri zed image analysis was used to determine the mean silver grain density in human kidney and bone cells following conventional in situ hybridi zation and after various cycles of IS-RT-PCR. Only a few cycles of PCR were needed to produce an optimum signal, but amplification of signal following IS-RT-PCR was found to be relatively inefficient. following the optimum number of cycles of IS-RT-PCR in kidney sections, there w as a less than four-fold increase in signal. Similarly, in bone, the o ptimum signal detected was only approximately five times greater than that found with conventional in situ hybridization. These results clea rly demonstrate that the increase in signal following IS-RT-PCR follow s a more linear pattern and is relatively inefficient, compared with t he usual exponential increase with conventional solution phase RT-PCR. (C) 1997 by John Wiley & Sons, Ltd.