PMID- 9774624
OWN - NLM
STAT- MEDLINE
DCOM- 19981103
LR  - 20170214
IS  - 0022-1554 (Print)
IS  - 0022-1554 (Linking)
VI  - 46
IP  - 11
DP  - 1998 Nov
TI  - Sensitive mRNA detection by fluorescence in situ hybridization using horseradish 
      peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification.
PG  - 1249-59
AB  - 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.
FAU - van de Corput, M P
AU  - van de Corput MP
AD  - Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, 
      Leiden University Medical Centre, Leiden, The Netherlands.
FAU - Dirks, R W
AU  - Dirks RW
FAU - van Gijlswijk, R P
AU  - van Gijlswijk RP
FAU - van Binnendijk, E
AU  - van Binnendijk E
FAU - Hattinger, C M
AU  - Hattinger CM
FAU - de Paus, R A
AU  - de Paus RA
FAU - Landegent, J E
AU  - Landegent JE
FAU - Raap, A K
AU  - Raap AK
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Histochem Cytochem
JT  - The journal of histochemistry and cytochemistry : official journal of the 
      Histochemistry Society
JID - 9815334
RN  - 0 (Cytokines)
RN  - 0 (Oligonucleotides)
RN  - 0 (RNA, Messenger)
RN  - 0 (biotinyltyramide)
RN  - 6SO6U10H04 (Biotin)
RN  - EC 1.11.1.- (Horseradish Peroxidase)
RN  - X8ZC7V0OX3 (Tyramine)
SB  - IM
MH  - Biotin/*analogs & derivatives
MH  - Blotting, Northern
MH  - Cytokines/metabolism
MH  - *Horseradish Peroxidase
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Oligonucleotides/*analysis
MH  - RNA, Messenger/metabolism
MH  - Reverse Transcriptase Polymerase Chain Reaction
MH  - Sensitivity and Specificity
MH  - Tumor Cells, Cultured
MH  - Tyramine/*analogs & derivatives
EDAT- 1998/10/17 00:00
MHDA- 1998/10/17 00:01
CRDT- 1998/10/17 00:00
PHST- 1998/10/17 00:00 [pubmed]
PHST- 1998/10/17 00:01 [medline]
PHST- 1998/10/17 00:00 [entrez]
AID - 10.1177/002215549804601105 [doi]
PST - ppublish
SO  - J Histochem Cytochem. 1998 Nov;46(11):1249-59. doi: 10.1177/002215549804601105.