Quantification of vitamin D receptor mRNA in tissue sections demonstrates 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 levelled at this technique is that positive results may be meaningless, as amplification is required to demonstrate the transcripts of interest. The use of IS-RT-PCR to demonstrate mRNA for receptors for 1,25-dihydroxyvitamin D3 (VDR) in sections of human kidney and bone has previously been described. To ascertain whether the levels of VDR mRNA detected following IS-RT-PCR were transcriptionally significant, computerized image analysis was used to determine the mean silver grain density in human kidney and bone cells following conventional in situ hybridization 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 was a less than four-fold increase in signal. Similarly, in bone, the optimum signal detected was only approximately five times greater than that found with conventional in situ hybridization. These results clearly demonstrate that the increase in signal following IS-RT-PCR follows a more linear pattern and is relatively inefficient, compared with the usual exponential increase with conventional solution phase RT-PCR.
Mee
A P
AP
University Department of Medicine, Manchester Royal Infirmary, U.K. andy.mee@man.ac.uk
Denton
J
J
Hoyland
J A
JA
Davies
M
M
Mawer
E B
EB
D001842:Bone and Bones / Q000378:metabolism*; D006801:Humans; D006962:Hyperparathyroidism, Secondary / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization / Q000379:methods*; D007668:Kidney / Q000378:metabolism*; D010001:Osteitis Deformans / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D018167:Receptors, Calcitriol