Abstract
BackgroundCurrent 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.ResultsThis 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.ConclusionThis method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.
Highlights
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 purified
Real-time polymerase chain reaction (PCR) in combination with reverse transcription (RT) provides a powerful tool for accurate quantification of DNA and RNA copy numbers and has opened the way to the study of subtle modulations of gene expression in small numbers of cells, as well as small-scale genetic analyses aimed at establishing chromosome numbers, the presence of mutations, or allele dropout
Single-tube Xist RNA, Xist DNA and Sry DNA quantification in individual male and female blastocysts The PurAmp method presented in this study is performed in a single tube from cell lysis to cDNA or genomic DNA amplification, eliminating possible loss of template molecules due to procedures such as phase separation and recovery, repeated washing and re-suspension of nucleic acid pellets, elution from binding matrices and vessel-tovessel transfer
Summary
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 purified. Real-time polymerase chain reaction (PCR) in combination with reverse transcription (RT) provides a powerful tool for accurate quantification of DNA and RNA copy numbers and has opened the way to the study of subtle modulations of gene expression in small numbers of cells, as well as small-scale genetic analyses aimed at establishing chromosome numbers, the presence of mutations, or allele dropout. The reliability of these measurements, depends on the accuracy of each step, including preparation and recovery of RNA and/or DNA, reverse transcription of RNA into cDNA, and quantifiable and specific amplification of all desired sequences. Mild detergents that do not lyse the nuclear membrane preclude quantification of DNA or RNA located in the nucleus, and are unlikely to completely remove proteins bound to cytoplasmic RNA
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