Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.
BMC Biotechnol
; 5: 2, 2005 Jan 13.
Article
em En
| MEDLINE
| ID: mdl-15649321
ABSTRACT
BACKGROUND:
Current methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost.RESULTS:
This paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3), as well as their RNA transcripts in single mouse embryos and in isolated blastomeres.CONCLUSION:
This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Blastocisto
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Biotecnologia
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DNA
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RNA
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Regulação da Expressão Gênica no Desenvolvimento
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Reação em Cadeia da Polimerase Via Transcriptase Reversa
Limite:
Animals
Idioma:
En
Ano de publicação:
2005
Tipo de documento:
Article