RiboGreen RNA Reagent

1. Overview
In RiboGreen's RNA quantification detection, two different dye concentrations are required to obtain a complete linear kinetic range. Before preparing the RiboGreen working solution (see reagent preparation below), it is necessary to decide whether to perform a high range assay (20ng/mL-500ng/mL RNA), a low range assay (1ng/mL-50ng/mL RNA), or both ranges.
2. Preparation of reagents
On the day of the experiment, RiboGreen's water-soluble working solution was prepared in a sterile disposable polypropylene plastic tube. Avoid using glassware as RiboGreen may adhere to glass surfaces. The prepared RiboGreen working solution should be stored away from light. It is best to use up the working fluid within a few hours after preparation, and do not store it for long-term use.This product is supplied as a stock solution dissolved in DMSO, with a concentration of 200x (High-range assay) and 2000x (Low-range assay). Based on a 2ml reaction system (cuvette), a 100μL sample can perform 20 reactions (High-range assay) and a 100μL sample can perform 200 reactions (Low-range assay).
3. Prepare RNA standard curve
For the standard curve, commercial RNA standards can be used, such as the commonly used 16S and 23S ribosomal RNA; Or other purified RNA products. Sometimes, it is recommended to use RNA standards similar to the sample being tested to establish a standard curve. Generally speaking, the vast majority of single stranded RNA molecules can produce almost equivalent fluorescence signals. In the presence of common compounds that contaminate nucleic acid reagents such as nucleosides, salts, urea, ethanol, chloroform, detergents, proteins, and agarose, RiboGreen's detection results can still maintain a good linear relationship, even if the fluorescence intensity may be affected . In order to ensure effective control, the RNA solution used to prepare the standard curve should be consistent with the sample to be tested, which can ensure the presence of impurity compounds at similar levels.
3.1 Prepare a 2 μ g/mL RNA solution in a plastic bottle using a 1xTE buffer. Measure the basic absorbance of the RNA solution at 260nm (A260) using a colorimetric cup in a 1cm long pathway. A260=0.05 is equivalent to a concentration of 2 μ g/mL of RNA A.
When preparing the High range standard curve, a series of 2x final concentration RNA standard solutions are prepared by diluting 2 μ g/mL RNA solution into a disposable nuclease free plastic tube, and finally transferred to a colorimetric dish or culture plate.
3.3 When preparing the Low Range standard curve, a series of 2x final concentration RNA standard solutions are prepared by diluting 2 μ g/mL RNA solution into a disposable nuclease free plastic tube, and finally transferred to a colorimetric dish or culture plate
3.4 Mix equal volumes of RiboGreen reagent working solution (see 2-Reagent Preparation) and 2xRNA standard solution. Incubate at room temperature in the dark for 2-5 minutes, ensuring that a sufficient volume of measuring solution is added to the measuring container. For a 10x10mm colorimetric dish, it should not be less than 2mL; For each hole of the 96 well plate, do not lower it than 200 μ L.
3.5 Choose to use a fluorescence spectrophotometer or fluorescence enzyme-linked immunosorbent assay (ELISA) reader to measure the fluorescence value of the sample, and select the standard fluorescence wavelength (excitation~480nm, emission~520nm).
3.6 Subtract the blank fluorescence value from the fluorescence value of each sample. Afterwards, use the calibrated fluorescence values as the y-axis and RNA concentration as the x-axis to establish a standard curve.
4. Sample analysis
4.1 Dilute the sample with a 1xTE buffer to an appropriate volume (e.g. 1.0mL to 10x10 mm cuvette or 100 μ L to 96 well microplate). It is best to do more than one dilution factor for each sample to be tested.
The higher the dilution factor of the test sample, the more helpful it is to reduce the interference of specific pollutants. However, it is also necessary to avoid particularly small sample volumes as they are difficult to measure accurately. In addition, in the same experiment, the impurity levels should be kept as consistent as possible to reduce the signal differences between samples caused by impurities. For example, if a series of RNA samples have significantly different salt concentrations, they cannot be compared using a simple standard curve. To avoid such problems, if possible, adjust the impurity concentration in all samples to the same level. (See 5- DNA clearance in sample)
4.2 Add an equal volume of RiboGreen measuring reagent (see 2-Reagent Preparation) to each sample and incubate at room temperature in the dark for 2-5 minutes.
4.3 Select the same instrument parameters as the standard curve measurement to determine the fluorescence value of the sample. In order to reduce the photobleaching effect, the fluorescence measurement time of all samples remains unchanged.
4.4 Subtract the blank fluorescence value from the fluorescence value of each sample. Determine the RNA concentration of the sample based on the generated RNA standard curve.
If possible, verify the quantitative results by diluting different samples and repeating the measurements.
5. DNA clearance in the sample
RiboGreen can also bind to DNA, and RNA free DNases can be used to remove DNA from the sample first, thereby avoiding fluorescence interference caused by the binding of DNA and RiboGreen, ensuring that all fluorescence in the sample comes from the binding of RiboGreen and RNA.
5.1 Prepare 10x DNase digestion buffer: 200mM Tris HCl without nuclease, pH 7.5,100 mM MgCl2,20 mM CaCl2。
5.2 Add 0.11 times the volume of 10xDNase digestion buffer to each DNA containing sample. (For example, if the sample is 9mL, add 1mL of 10x buffer).
5.3 Add~5 units of DNase without RNase to 1 μ g of DNA.
5.4 Incubate at 37 ° C for 90 minutes.
5.5 Dilute the sample at least 10 times with a 1xTE buffer to reduce interference from residual salt ions in the digestion solution on RiboGreen measurements.
5.6 Perform RiboGreen assay experiment according to the above steps.