Multiple molecular analyses from minimal cell quantities by sequential isolation and preamplification of DNA and RNA.
Molecular analyses of single or very few cells are often handicapped by low amounts of DNA or RNA from starting material. Specimens like biopsies, embryonic stem cells, or laser-microdissected tissues often do not provide nucleic acid quantities required for robust amplification and verification by diagnostic polymerase chain reaction (PCR)-based analyses. A simultaneous isolation procedure for both RNA and DNA from a single sample would greatly facilitate demanding molecular studies. Here, we describe a new method that combines the isolation of mRNA using commercially available oligo (dT)-bound magnetic beads with the precipitation of DNA from the remaining cell lysate. Both types of nucleic acids can then be separately preamplified by improved primer extension preamplification and subsequently used for multiple molecular analyses. With this procedure we were able to reliably gain DNA for PCR from as few as 5 cells. RNA suitable for various reverse transcription-PCR approaches and sequencing analyses could simultaneously be isolated even from single cell preparations.
Koehler
Astrid
A
Department of Pathology and Molecular Diagnostics, University of Regensburg, Germany.
Kunz-Schughart
Leoni A
LA
Hofstaedter
Ferdinand
F
Dietmaier
Wolfgang
W
D001483:Base Sequence; D045744:Cell Line, Tumor; D004247:DNA / Q000302:isolation & purification*; D017931:DNA Primers; D006801:Humans; D018189:Immunomagnetic Separation; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; C027903:oligo (dT); D004247:DNA