RNA的质量和纯度鉴定VIP免费

RNA 的质量和纯度鉴定2011 年 02 月 22 日 星期二 04:17摘 自RNA: A Laboratory Manual, by Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, andTimothy W.Nilsen. CSHL Press, Cold Spring Harbor, NY,USA, 2010.RELATED INFORMATIONSeveral methods for RNA purification are described in Purification of RNA by SDS Solubilizationand Phenol Extraction (Rio et al. 2010a) and Ethanol Precipitation of RNA and the Use of Carriers(Rio et al. 2010b). RNA samples contaminated with DNA can be purified using DNAse I, asdescribed in Removal of DNA from RNA (Rio et al. 2010c). RNA molecules ≤600 kb can beanalyzed using polyacrylamide gel electrophoresis (Polyacrylamide Gel Electrophoresis of RNA[Rio et al. 2010d]), whereas larger RNA molecules such as messenger RNA (mRNA) are moreeffectively analyzed on agarose gels (Nondenaturing Agarose Gel Electrophoresis of RNA [Rio etal. 2010e]). A protocol is also available for Northern Hybridization (Sambrook and Russell 2006).Before attempting to use any of the following procedures for DNA quantitation, it is important tohave a "ballpark" idea of what you expect the yield to be (see Table 1).DETERMINING YIELD BY SPECTROPHOTOMETRY OR FLUORIMETRYThe easiest way to determine the quantity of RNA in a sample is to measure the absorbance at260 nm (A260) using a spectrophotometer. Because the bases in RNA absorb ultraviolet (UV) lightin the 250- to 265-nm range, one can use this property to quantitatively measure theconcentration of an RNA solution, using an average absorbance for the four nucleotide bases. Asolution of RNA at 40 µg/mL will have an absorbance of ~1. Accordingly, if 50 µL of the samesolution is diluted in 1 mL of H2O and read in a 1-mL cuvette, the absorbance will be 0.05;...