PMID- 9819811
OWN - NLM
STAT- MEDLINE
DCOM- 19990120
LR  - 20230315
IS  - 1073-6085 (Print)
IS  - 1073-6085 (Linking)
VI  - 10
IP  - 2
DP  - 1998 Oct
TI  - Quantitative aspects of an in situ hybridization procedure for detecting mRNAs in 
      cells using 96-well microplates.
PG  - 107-13
AB  - The universal quantitation of the DNA hybridization reaction has been a goal 
      sought by many researchers. Part of this search has been the need to develop a 
      rapid, sensitive, easy-to-perform, and quantitative method to measure the 
      abundance of specific mRNAs directly within cells. Conventionally mRNA detection 
      can be done by advanced quantitative in situ hybridization (ISH) using either 
      image analysis or fluorescence in situ hybridization (FISH), or indirectly by 
      extraction of mRNA from cells or tissue and using Northern blot or quantitative 
      polymerase chain reaction (PCR). We examined the quantitative nature of probe 
      binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method 
      of ISH previously developed in our laboratories. The method is applicable to 
      isolated primary cells or cells in culture. The procedural details are very 
      simple, with cells being centrifuged into 96-well microplates, fixed with 
      formalin, and pretreated with Triton X-100 and Nonidet P-40 before 
      photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of 
      probe to target is detected using multiple applications of streptavidin and 
      biotinylated alkaline phosphatase and visualized by the p-nitrophenyl phosphate 
      conversion method. The quantitative parameters of the ISH procedure were 
      determined by measuring the levels of expression of erythropoietin (EPO) mRNA and 
      its translated protein in transfected COS-7 cells. There is a log-linear 
      relationship between the levels of signal obtained in the ISH reaction in 96-well 
      microplates and the EPO protein levels measured by enzyme-linked immunosorbent 
      assay (ELISA). This demonstrated relationship is important in the standardization 
      and use of these procedures to measure quantitatively mRNAs within cells.
FAU - Zreiqat, H
AU  - Zreiqat H
AD  - Bone Biomaterials Unit, University of New South Wales, Sydney, Australia.
FAU - Sungaran, R
AU  - Sungaran R
FAU - Howlett, C R
AU  - Howlett CR
FAU - Markovic, B
AU  - Markovic B
LA  - eng
PT  - Journal Article
PL  - Switzerland
TA  - Mol Biotechnol
JT  - Molecular biotechnology
JID - 9423533
RN  - 0 (RNA, Messenger)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 11096-26-7 (Erythropoietin)
SB  - IM
MH  - Animals
MH  - COS Cells/cytology/metabolism
MH  - Enzyme-Linked Immunosorbent Assay
MH  - Erythropoietin/genetics
MH  - Genetic Vectors/genetics
MH  - In Situ Hybridization/instrumentation/*methods
MH  - Plasmids/genetics
MH  - RNA, Messenger/*analysis/genetics
MH  - Recombinant Fusion Proteins/genetics
EDAT- 1998/11/20 00:00
MHDA- 1998/11/20 00:01
CRDT- 1998/11/20 00:00
PHST- 1998/11/20 00:00 [pubmed]
PHST- 1998/11/20 00:01 [medline]
PHST- 1998/11/20 00:00 [entrez]
AID - 10.1007/BF02760859 [doi]
PST - ppublish
SO  - Mol Biotechnol. 1998 Oct;10(2):107-13. doi: 10.1007/BF02760859.