Nucleic acids are accurately quantified using spectrophotometry because this method does not destroy the structure and also allows for sample recovery. RNA has the property of absorbing ultraviolet light with a peak absorption at 260nm, which is the average of the absorption values of individual ribonucleic acids between 256nm and 281nm.
Operation method
RNA spectroscopy and quantification
Materials and Instruments
DEPC Nuclease-free water Move I. Materials and equipment Caveat 1) Gloves should be worn throughout the procedure to minimize the effect of exogenous RNase activity.2) It is often recommended to analyze RNA by gel electrophoresis rather than spectrophotometry because electrophoresis not only reveals the integrity of the RNA, but also, in most cases, the type of RNA, e.g., 18S/23SrRNA in prokaryotic cells and 18S/28SRNA in eukaryotic cells.3) The concentration of RNA can be roughly determined by analyzing the bands with EB.4) In addition, RNA samples can be analyzed for quantification and integrity by fluorescent staining and the Agilent 2100 Bioanalyzer. Some fluorescent dyes, such as RiboGreen, increase the fluorescence intensity significantly after binding to nucleic acids, and RNA at a concentration of lng/ml can be detected and quantified by the fluorescence analyzer after staining with RiboGreen. For accurate quantification of RNA, a standard curve can be set up with a sample of known concentration, and when two different dye concentrations are used, the linear range of the RiboGreen assay includes three orders of magnitude (lng/ml to lug/ml). Furthermore, the RiboGreen method is relatively insensitive to non-nucleic acid substances in the sample, so the _concentration is linearly related to the absorbance value. The following points should be noted when using this method: first, RiboGreen cannot distinguish between DNA and RNA, so DNA should be removed with RNase-free DNase; second, RiboGreeii adsorbs to the wall of the tube, so non-adsorbing, nuclease-free polypropylene tubes should be used; and third, in order to avoid photodegradation of the RiboGreeii reagent, the reagent should be stored away from light, and the reagent should be stored at a low temperature after preparation. Third, to avoid photodegradation of RiboGreeii reagents, they should be stored out of the light and used within a few hours of preparation; finally, repeated freezing and thawing of RNA standards should be avoided to avoid shearing of the RNA strand and thus decreasing the ability to bind the dye. Finally, repeated freezing and thawing of RNA standards should be avoided, as this may cause shearing of the RNA strands and reduce the ability to bind to the dye.5) Agilent2100 Bioanalyzer combines capillary electrophoresis, fluorescence staining and other technologies to quantify and analyze the integrity of RNA □ Its biggest advantage lies in the analysis of RNA integrity, which can display the peak and ratio of 18S and 2SS ribosomal RNA; in addition, it is also very characteristic for the analysis of the integrity of mRNA and aRNA (tantisenseRNA). The integrity analysis of mRNA and aRNA (tantisenseRNA) is also very characteristic. The integrity of mRNA and aRNA mainly consists of bands ranging from lkb to 2kb, and the presence of more low molecular mass bands implies that the integrity of RNA is poor. For more product details, please visit Aladdin Scientific website.
UV spectrophotometer Quartz colorimetric cups
1) Ultraviolet spectrophotometer.
2) Quartz colorimetric cup.
3) DEPC
4) Nuclease free water.
II. Method of operation
(i) Preparation of spectrophotometer
Soak the cuvette in 0.1% DEPC water for at least 15 min.
2) Set up the baseline with water or buffer tuck without UV absorption.
3) Dilute RNA samples with water or non-UV absorbing buffer and perform assay.
4) Record the absorption values of the samples at 230nm, 260nm and 280nm, and print the scanning curves at 200~300nm.
(ii) Analysis of spectrophotometric scanning curve
In some cases, it is necessary to understand the degree of contamination by scanning analysis rather than just measuring the absorption values at 260nm and 280mn. After extraction of residual trace phenol, guanidine thiocyanate, the scanning graphs are significantly different, A260: A280 values will be slightly different, m is still within the acceptable range, and the RNA concentration will be a significant change.
Therefore, when analyzing the RNA ratio and concentration by spectrophotometry, not only the absorption values of 280nm, 260nm and 230nm should be considered, but also the scanning in the range of 200~300nm. The trace residues of reagents used in the process of RNA extraction can affect and mislead the results of the determination, and then affect the subsequent experiments.
(C) RNA quantification
Train RNA samples in the 260mn at the light absorption value and sample dilution times, UV spectrophotometer can be measured directly out of the concentration of the sample, but also can be calculated according to the following formula: RNA concentration (ug/ml) = ( A260X dilution times) / (0.024XL) where: A260 for the 260mn wavelength at the light absorption value; L is the thickness of the colorimetric cell, generally 1 cm or 0.5 cm; 0.024 for each millimeter of light absorption value; L is the thickness of the colorimetric cell, generally 1 cm or 0.5 cm; 0.024 for each millimeter of the colorimetric cell, generally 1 cm or 0.5 cm; 0.024 for each millimeter of the RNA quantitative analysis. L is the thickness of the colorimetric cell, usually 1 cm or 0.5 cm; 0.024 is the light absorption value of 1ugRNA per milliliter of solution. When the thickness of the colorimetric cell is lcm and the A260 of the sample is 1, the concentration of RNA is about the dilution factor X40ug/ml.