Single-cell cDNA-PCR: removal of contaminating genomic DNA from total RNA using immobilized DNase I.
A procedure utilizing immobilized DNase I that allows the efficient amplification of cDNA by PCR from a single cell in the absence of contaminating genomic DNA is described. DNase I treated, total RNA derived from single cells was reverse transcribed into cDNA followed by PCR using beta-actin and c-fos specific primers that recognize different exons of the respective genes. Amplification products corresponding to cDNA, but not to genomic sequences, were detected after treatment with immobilized DNase I in samples previously shown to be contaminated with genomic DNA. This method allows the efficient removal of DNA contaminating total RNA derived from a single cell.
Ziegler
B L
BL
Dept. of Clinical Physiology & Occupational Medicine, University of Ulm, FRG.
Lamping
C
C
Thoma
S
S
Thomas
C A
CA
D016477:Artifacts; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D003850:Deoxyribonuclease I; D004800:Enzymes, Immobilized; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*
D004800:Enzymes, Immobilized; D012333:RNA, Messenger; D004247:DNA; D003850:Deoxyribonuclease I