Modification of the Standard Trizol-Based Technique Improves the Integrity of RNA Isolated from RNase-Rich Placental Tissue
2006; American Association for Clinical Chemistry; Volume: 52; Issue: 1 Linguagem: Inglês
10.1373/clinchem.2005.059758
ISSN1530-8561
AutoresRonit Haimov‐Kochman, Susan J. Fisher, Virginia D. Winn,
Tópico(s)RNA Research and Splicing
ResumoPreservation of RNA integrity is important in microarray techniques for identifying differentially expressed genes so that results reflect true biological differences and not differences in RNA degradation (1). RNA degradation is usually low in RNA isolated from cultured cells (2). When samples isolated from RNase-rich tissues are used, however, RNA degradation may introduce bias (3)(4). RNA extraction using Trizol (Invitrogen Life Technologies) is a common procedure in microarray experiments. Tissue homogenization at room temperature (15–25 °C), as specified in the manufacturer’s protocol, generates heat that may increase RNase activity. When snap-frozen tissue pieces are processed, only the outer surfaces are initially in contact with Trizol, and RNase activity in the deeper regions could adversely affect RNA integrity. We modified the Trizol protocol by ( a ) performing homogenization of frozen placental specimens in cold Trizol on wet ice (0–4 °C), ( b ) limiting homogenization time to 30-s intervals for up to 1.5 min to minimize heat generation at the expense of complete tissue disruption, and ( c ) pelleting cellular debris at 12 000 g for 10 min at 4 °C rather than at room temperature (see the Supplemental Methods section in the Data Supplement that accompanies the online version of this Letter at http://www.clinchem.org/content/vol52/issue1/). Using capillary electrophoresis [RNA …
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