Preparation of both DNA and RNA for hybridization analysis from limiting quantities of lymphoid cells

• Published in: Immunology letters Vol. 18; no. 3; pp. 219 - 223
• Main Authors: Pearse, Martin J., Wu, Li
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.07.1988; Elsevier
• Subjects: Animals; Biological and medical sciences; Cell Count; Cell Fractionation - methods; Diverse techniques; DNA - isolation & purification; Fundamental and applied biological sciences. Psychology; Gel electrophoresis; Mice; Molecular and cellular biology; Northern blotting; Nucleic Acid Hybridization; Nucleic acids; RNA - isolation & purification; Southern blotting; T-Lymphocytes - analysis; Northern blotting; Nucleic acids; Southern blotting; Gel electrophoresis; Lymphoid cell; Purification; Method; RNA; DNA; Nucleic acid
• ISSN: 0165-2478; 1879-0542
• DOI: 10.1016/0165-2478(88)90022-3

This report describes a method for preparing both DNA and RNA simultaneously from as few as 5 × 10 5 lymphoid cells. The method is suitable for cultured cells or any tissue from which a cell suspension can be prepared and for the small samples of purified cells obtained by fluorescence activated cell sorting. Cells are lysed with Nonidet P-40 and the nuclear and cytoplasmic fractions separated by centrifugation. Nuclei are embedded in low-gelling-temperature agarose and the proteinase K and restriction enzyme digestions performed whilst the DNA is immobilized in this form. Total RNA is prepared from the cytoplasmic fraction. This method is simple but reliable and is therefore particularly useful for preparing and analyzing the DNA and RNA from multiple samples when material is limited.