Hi,

I can imagine several reasons. First, make sure that your device is calibrated and/or the baseline could have been incorrectly set.

Other explanation can be that the RNA concentration is extremely low and therefore, the absorbance might be close to or below the detection limit of the spectrophotometer.

Hello Aya Youssef

The uv spectro shows relatively low sensitivity and selectivity. It may thus be difficult to detect very low concentration of an analyte in the sample.

Below are some of the factors that may be considered.

1. Sample (RNA) readings should be taken in quartz cuvette. Dirty cuvettes and dust particles cause light scatter at 320nm which can impact absorbance at 260nm.

2. Make sure that you blank the spectrophotometer with the same buffer that is used for the sample before taking any measurements because this ensures the absorbance measured in the sample is attributed only to the sample material and not the components of the solution the sample is suspended in or the cuvette.

3. Make sure your RNA dilution is within the linear range of your spectrophotometer. Usually, absorbance value should fall between 0.1 and 1.0. Solutions that are outside this range cannot be measured accurately. Generally greater error occurs at lower concentrations. The use of 5ul RNA sample may be too low. You may have to increase the volume of your sample.

4. If you get the A260 absorbance of your sample as 0.1, which corresponds to 4ug/ml RNA, it is often impractical to use uv spectro to quantitate RNA isolated from samples that will have lower concentration once diluted. You should use alternate methods to accurately quantify small amounts of RNA such as the fluorescent dye-based quantification which involves the use of fluorescent dyes that bind to RNA. In this method, a nucleic acid sample along with a series of standards of known concentrations are incubated with the fluorescent dye. The dye binds to the nucleic acid and undergoes a conformational change, resulting in increased fluorescence at a wavelength specific to the dye being used. Fluorescence is measured, and a standard curve is created by plotting fluorescence against nucleic acid concentrations of the known standards. The fluorescence of the unknown sample then is converted to a nucleic acid concentration using the linear regression equation that best describes the standard curve.

Best.

your device isnt calibrated.

Also, the quality of the water added to the sample and the water used for calibration And zeroing the device may have problems.