Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.
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.


CONCLUSIONS
This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.
Hartshorn
Cristina
C
Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA. hartcris@brandeis.edu
Anshelevich
Aleksandra
A
Wangh
Lawrence J
LJ
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D001755:Blastocyst / Q000166:cytology*; D004247:DNA / Q000032:analysis*; D003851:Deoxyribonucleases / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012313:RNA / Q000032:analysis* / Q000235:genetics; D062085:RNA, Long Noncoding; D012333:RNA, Messenger / Q000378:metabolism; D022661:RNA, Untranslated / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D051876:Sex-Determining Region Y Protein / Q000235:genetics; D013997:Time Factors; D014158:Transcription, Genetic
D018840:HSP70 Heat-Shock Proteins; C498550:Hsp70.3 protein, mouse; D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D022661:RNA, Untranslated; D051876:Sex-Determining Region Y Protein; C497746:Sry protein, mouse; C077885:XIST non-coding RNA; C081884:heat-shock protein 70.1; D012313:RNA; D004247:DNA; D003851:Deoxyribonucleases