Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction.
Human epidermal growth factor (EGF) receptor mRNA was detected in cryopreserved tissue sections adherent to whole glass slides using in situ reverse transcriptase polymerase chain reaction. EGF receptor cDNA was synthesized in situ by reverse transcription using an EGF receptor-specific oligonucleotide primer. In situ polymerase chain reaction amplification in the presence of digoxigenin-11-dUTP and subsequent binding with an antidigoxigenin antibody conjugated to alkaline phosphatase allowed direct visualization. Because DNase, RNase, or proteinase K are not required, tissue integrity is maintained. EGF receptor mRNA is expressed in the basal layer of normal human skin epithelium and is significantly overexpressed in squamous cell tumor specimens, which is consistent with conventional analysis of EGF receptor expression. The assay is semiquantitative, quicker, more sensitive, and void of the nonspecific binding associated with in situ hybridization. In situ reverse transcriptase polymerase chain reaction using whole glass slides is ideally suited for detecting moderate to infrequently expressed transcripts in biopsy specimens.
Patel
V G
VG
Department of Surgery, University of Louisville, Kentucky.
Shum-Siu
A
A
Heniford
B W
BW
Wieman
T J
TJ
Hendler
F J
FJ
D016375:Antisense Elements (Genetics) / Q000235:genetics; D001483:Base Sequence; D001706:Biopsy; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology; D018076:DNA, Complementary / Q000032:analysis; D066246:ErbB Receptors / Q000235:genetics*; D006801:Humans; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D012867:Skin / Q000378:metabolism / Q000473:pathology
D016375:Antisense Elements (Genetics); D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D066246:ErbB Receptors; D012194:RNA-Directed DNA Polymerase