RNA Cell HTS 96 Kits

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AppendixGeneral notes on handling RNARNA is far less stable than DNA. It is very sensitive to degradation by endogenous RNases in thebiological material and exogenous RNases which are permanently present everywhere in the lab.To achieve satisfactory qualitative and quantitative results in RNA preparations, contaminationswith exogenous RNases has to be reduced as much as possible Avoid handling bacterial cultures,cell cultures or other biological sources of RNases in the same lab where the RNA purification is tobe carried out.All glassware should be treated before use to ensure that it is RNase-free. Glassware should becleaned with detergent, thoroughly rinsed and oven baked at 240°C for four or more hours beforeuse. Autoclaving alone will not completely inactivate many RNases. Oven baking will bothinactivate RNases and ensure that no other nucleic acids (such as Plasmid DNA) are present onthe surface of the glassware. You can also clean glassware with 0.1% DEPC (diethylpyrocarbonate). The glassware must stand 12 hours at 37°C and then be autoclaved or heated to100°C for 15 min to remove residual DEPC○○○○○○○○○○○○electrophoresis tanks should be cleaned with detergent solution (e.g. 0.5% SDS), thoroughlyrinsed with RNase-free water, and then rinsed with ethanol and allowed to drynon-disposable plasticware should be treated before use to ensure that it is RNase-free.Plasticware should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA followed by RNasefreewater. You can also take chloroform-resistant plasticware rinsed with chloroform toinactivate RNasesall buffers must be prepared from DEPC-treated RNase-free ddH 2 Owhen working with chemicals, always wear a suitable lab coat, disposable gloves, andprotective goggleschange gloves frequently and keep tubes closedall centrifugation steps are carried out at room temperatureto avoid cross contamination cavity seams shouldn’t be moisted with fluidreduce the preparation time as much as possibleuse only sterile, disposable polypropylene tubes throughout the procedure. (these tubesare generally RNase free)keep isolated RNA on icedo not use kit components from other kits with the kit you are currently using, unless the lotnumbers are identicalto minimize the risk of infections from potentially infectious material, we recommend workingunder laminar air-flow until the samples are lysedThis kit should only be used by personnel trained in in vitro diagnostic laboratory practiceStorage of RNAPurified RNA can be stored –80°C and is stable for months and years, e.g. precipated and storedunder 70% ethanol.Quantification of RNAThe concentration of RNA should be determinate by measuring the absorbance at 260 nm (A 260 ) inphoto spectrometer. Readings should be greater than 0.15 to ensure significance. An absorbanceof 1 unit at 260 nm corresponds to 40 µg of RNA per ml. This relation is valid only formeasurements at neutral pH. The ratio between absorbance values at 260 nm and 280 nm givesan estimate of RNA purity (see below)21 InviTrap ® RNA Cell HTS 96 Kit/ C and V 0810
When measuring RNA samples, make sure that cuvettes are RNase-free, esp. if the RNA is to berecovered after photo spectrometry. Thioas can be accomplished by washing cuvettes with 0.1NaOH, 1 mM EDTA folloed by washing with RNase-free water. Use buffer in which the RNA isdiluted to zero the photo spectrometer.An example of the calculation involved in RNA quantification:Volume of RNA sample 100 µlDilution = 20 µl of RNA sample + 180 µl of 10 mM Tris, pH 7.0 (1/10 dilution).Measure absorbance of diluted sample in a 0.2 ml cuvette (RNase free)A 260 = 0.2Concentration of RNA sampleTotal amount= 40 µg/ml * A 260 * dilution factor= 40 µg/ml * 0.2 * 10= 80 µg= concentration * volume of sample in ml= 80 µg/ml * 0.1 ml= 8 µg of RNAPurity of RNAThe ratio of the readings at 260 nm and 280 nm (A 260 /A 280 ) provides an estimate of the purity ofRNA with respect to the contaminants that absorb in the UV, such as protein. However, theA 260 /A 280 ratio is influenced considerably by pH. Since water is not buffered, the pH and theresulting A 260 /A 280 ratio can vary greatly. Lower pH results in lower A 260 /A 280 ratio and reducedsensitivity to protein contamination.* For accurate values, it is recommend to measure absorbancein 10 mM Tris Cl, pH 7.5. Pure RNA has an A 260 /A 280 ratio of 1.9-2.1** in 10 mM Tris Cl, pH 7.5.Always be sure to calibrate the spectrophotometer with the same solution.For determination of RNA concentration, however, it is recommend to dilute the sample in a bufferwith neutral pH since the relationship between absorbance and concentration (A 260 reading of 1 =40 µg/ml of RNA) is based on an extinction coefficient calculated for RNA at neutral pH.* Wilfinger, W.W.,Mackey, M., and Chomczynski, P. (1997) Effect of Ph and ionic strength on the spectrophotometricassessment of nucleic acid purity. Bio Techniques 22, 474** Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris Cl, pH 7.5) with some photo spectrometers22 InviTrap ® RNA Cell HTS 96 Kit/ C and V 0810
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