Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification.
With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1alpha and beta-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods. Another method is the visualization of transcripts in tissue or cell cultures by fluorescence in situ hybridization (FISH). However, for low-abundance RNA detection, this method is hampered by its limited detection sensitivity and by the interference of background signals with specific hybridization signals. Background signals are introduced by nonspecific hybridization of probe sequences or nonspecific binding of antibodies used for visualization. To eliminate background signals derived from both sources and to benefit from the peroxidase-driven tyramide signal amplification (TSA), we directly conjugated horseradish peroxidase (HRP) to oligodeoxynucleotides (ODNs) and used these probes to study in the bladder cancer cell line 5637 the expression of various cytokine genes which, according to Northern hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) assays, are expressed at levels up to 10,000-fold less than abundantly expressed housekeeping genes. The results show that reduction of probe complexity and the limited use of immunocytochemical detection layers strongly reduces noise signals derived from nonspecific binding of nucleic acid probe and antibodies. The use of the HRP-ODNs in combination with TSA allowed detection of low-abundance cytokine mRNAs by FISH.
Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, The Netherlands.
M P
MP
van de Corput
R W
RW
Dirks
R P
RP
van Gijlswijk
E
E
van Binnendijk
C M
CM
Hattinger
R A
RA
de Paus
J E
JE
Landegent
A K
AK
Raap
D001710:Biotin / Q000031:analogs & derivatives*; D015152:Blotting, Northern; D016207:Cytokines / Q000378:metabolism; D006735:Horseradish Peroxidase; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009841:Oligonucleotides / Q000032:analysis*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014439:Tyramine / Q000031:analogs & derivatives*
D016207:Cytokines; D009841:Oligonucleotides; D012333:RNA, Messenger; C111551:biotinyltyramide; D001710:Biotin; D006735:Horseradish Peroxidase; D014439:Tyramine