Novel and sensitive detection systems for the vitamin D receptor--in situ-reverse transcriptase-polymerase chain reaction and immunogold cytochemistry.
The receptor for the active metabolite of vitamin D, 1,25(OH)(2)D(3), known as the vitamin D receptor (VDR), belongs to the steroid hormone nuclear receptor superfamily. We have developed novel methods for detection of VDR mRNA and protein within a human promyelomonocytic cell line, HL-60. Using the newly developed technique of in situ-reverse transcriptase-polymerase chain reaction (IS-RT-PCR), low levels of VDR mRNA could be amplified and demonstrated unequivocally within these cells, and also within a human kidney proximal tubule cell line, CL-8. Use of a novel immunogold cytochemical technique has allowed clear and sensitive detection of VDR protein expression within the HL-60 cells. Further development of IS-RT-PCR has allowed us to apply this technique to tissue sections. We have shown clear amplification of VDR transcripts within sections of formalin-fixed paraffin-embedded human kidney and liver. These techniques will be useful to localise specifically the VDR within cell types that contain low levels of mRNA and protein, and will permit further investigation of the role played by 1,25(OH)(2)D(3) in cellular regulatory mechanisms.
Mee
A P
AP
Bone Disease Centre, University Department of Medicine, Manchester Royal Infirmary, UK.
Davenport
L K
LK
Hoyland
J A
JA
Davies
M
M
Mawer
E B
EB
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods* / Q000706:statistics & numerical data; D017403:In Situ Hybridization / Q000379:methods / Q000706:statistics & numerical data; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D018167:Receptors, Calcitriol / Q000032:analysis* / Q000235:genetics*; D012680:Sensitivity and Specificity; D014018:Tissue Distribution
D017931:DNA Primers; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D012194:RNA-Directed DNA Polymerase