Quality assessment of total RNA英文资料.pdfVIP

Quality assessment of total RNA (from /index.html) • RNA quality control: NanoDrop® ND-1000 • RNA quality control: Agilents 2100 Bioanalyzer RNA quality control using the NanoDrop ND-1000 There are three quality controls that are performed on isolated RNA. One is to determine the quantity of RNA that has been isolated, the second is the purity of RNA that has been isolated and the third is the integrity of the RNA that has been isolated. Measuring the Quantity of RNA using the Nanodrop. Nucleic acids are traditionally quantified using UV absorption using a spectrophotometer. In its simplest form the absorbance is measured at 260 and 280 nm. The concentration of nucleic acid can be determined using the Beer-Lambert law, which predicts a linear change in absorbance with concentration. An A260 reading of 1,0 is equivalent to about 40 µg/ml of RNA and the OD at 260 nm is used to determine the RNA concentration in a solution. RNA has its absorption maximum at 260 nm and the ratio of the absorbance at 260 and 280 nm is used to assess the RNA purity of an RNA preparation. Pure RNA has an A260/A280 of 2,1. You will see in many protocols that a value of 1,8-2,0 indicates that the RNA is pure. This depends, however on how you performed the measurement and the source of putative contaminations. Ideally, scanning spectrophotometry should be used as this makes it possible to also identify possible sources of contamination. One of the problems using conventional spectrophotometers is that the cuvettes are large, making it difficult to measure low concentrations of RNA without loosing an unacceptable fraction of the sometimes precious and valuable RNA sample. The NanoDrop® ND-1000 UV-Vis Spectrophotometer enables highly accurate analyses of extremely small samples with remarkable reproducibility. The sample rete