Guidelines for submitting RNA

The concentration of RNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. To ensure significance, readings should be greater than 0.15 and less than 1. An absorbance of 1 unit at 260 nm corresponds to 40 µg of RNA per ml. Use the buffer in which the RNA is diluted to zero the spectrophotometer. An example of the calculation involved in quantifying RNA is shown below:

Volume of RNA sample = 1.6 ml

Dilution = 10 µl of RNA sample + 490 µl distilled water (1/50 dilution)

Measured absorbance (A260) of diluted sample in a 1 ml cuvette (RNase-free):

= 0.75

Concentration of RNA sample:

= 40 x A260 x dilution factor
= 40 x 0.75 x 50
= 1500 µg ml-1

Total yield (concentration x volume of sample in milliliters):

= 1500 µg ml-1 x 1.6 ml
= 2400 µg = 2.4 mg RNA

 

The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV, such as protein and phenol. However, the A260/A280 ratio is influenced considerably by pH and tells the user nothing regarding the integrity of the RNA sample. If there is contamination with protein or phenol, the A260/A280 will be significantly less than the values given above, and accurate quantitation of the amount of RNA will not be possible.

Note: Removal of contaminating genomic DNA by DNase treatment is not necessary for microarray analysis, and may result in a decrease in RNA integrity. However, if the sample is to be used for subsequent RT-PCR verification, to prevent any interference by DNA in RT-PCR applications, design primers that anneal at intron splice junctions so that genomic DNA will not be amplified. For sensitive applications, such as differential display, or if it is not practical to use splice-junction primers, DNase digestion of the purified RNA with RNase-free DNase is recommended.