|
Technical support : Extraction & purification
The extraction and the purification of RNA from biological samples is a critical step in a microarray experiment since it directly conditions the quality of the results.
Extract RNA as quickly as possible after obtaining samples to minimize the degradation of RNA by limiting the activity of endogenous RNAses, and to limit the biosynthesis of new RNA molecules that may both modify the gene expression profiles. For best results, use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at -70°C. As an alternative approach, biological samples can be stabilized with commercially available RNA-stabilisation reagents (RNAlater, Ambion, cat#7020).
A rapid purification of total RNA from cells and tissues can be achieved by acid phenol-Guanidinium Thiocyanate-chloroform extraction according to the method described by Chomczynski and Sacchi (Anal Biochem (1987), 162 : 156) or using a commercial ready-to-use reagent derived from this method (TRIzol Reagent, Invitrogen, cat#15596-026; RNA Now, Zyme, cat#CBX-101).
Poly(A)RNA are isolated from total RNA fractions by affinity chromatography using commercially available kits (Micro FastTrack 2.0 kit, Invitrogen, cat#K1520-02; mTRAP, Active motif, cat#23024).
It is strongly recommended to check poly(A)RNA preparation quantity, integrity and purity by UV spectrophotometry and agarose gel electrophoresis after denaturation with glyoxal (Sambrook et al., eds (1989) Molecular cloning - a laboratory manual, 2nd ed. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory Press). To save time and money, do not use any sample that shows some evidence of degradation or contamination since it probably won't give any reliable results. For pure RNA, the A260/A280 ratio is >= 2. A ratio smaller than 2.0 means that the preparation is contaminated with proteins or aromatic substances such as phenol. In this case, it is recommended to purify RNA again. After electrophoresis of total RNA extracted from mammalian cells, the 28s and 18s species of ribosomal RNA (rRNA), as well as a fast migrating band composed of transfer RNA and 5s rRNA should be clearly visible in the presence of ethidium bromide under UV illumination. If the RNA preparation is undegraded, no smearing of either band should be visible, and the 28s rRNA band should be stained at approximately twice the intensity of the 18s band. mRNA is not visible unless the gel is overloaded : it is a smear. Staining close to the loading well is indicative of the presence of contaminant DNA.
RNA can be safely stored aliquoted either in an aqueous buffer at -80°C (TE, 0.1-0.5% SDS pH 7.6 or 0.1 mM EDTA in DEPC-treated water pH 7.5), in deionized formamide at -20°C, or as a suspension in 75% Ethanol at -20°C.
<<
| >>
|