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Titolo:
Direct in situ reverse transcriptase-polymerase chain reaction
Autore:
Kher, R; Bacallao, R;
Indirizzi:
Indiana Univ, Sch Med, Richard L Roudebush Vet Affairs Med Ctr, Div Nephrol & Hypertens, Indianapolis, IN 46202 USA Indiana Univ Indianapolis IN USA46202 ertens, Indianapolis, IN 46202 USA
Titolo Testata:
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
fascicolo: 2, volume: 281, anno: 2001,
pagine: C726 - C732
SICI:
0363-6143(200108)281:2<C726:DISRTC>2.0.ZU;2-P
Fonte:
ISI
Lingua:
ENG
Soggetto:
RECEPTOR MESSENGER-RNA; TISSUE-SECTIONS; INSITU HYBRIDIZATION; RT-PCR; CELLS; DNA; AMPLIFICATION; LOCALIZATION; EXPRESSION; SPECIMENS;
Keywords:
deoxyribonuclease I; restriction enzymes;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
36
Recensione:
Indirizzi per estratti:
Indirizzo: Bacallao, R Indiana Univ, Sch Med, Richard L Roudebush Vet Affairs Med Ctr, Div Nephrol & Hypertens, Indianapolis, IN 46202 USA Indiana Univ Indianapolis IN USA 46202 anapolis, IN 46202 USA
Citazione:
R. Kher e R. Bacallao, "Direct in situ reverse transcriptase-polymerase chain reaction", AM J P-CELL, 281(2), 2001, pp. C726-C732

Abstract

In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.

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Documento generato il 26/01/20 alle ore 16:07:44