Checking the quality of total RNA by Spectrophotometry

IMPORTANT: The Center for Medical Genomics has found that using A260/A280 ratios is not adequate to judge either quality or quantity of total RNA! We have seen many samples with good A260/280 ratios in which the actual amount of RNA is only 10-20% of the nominal amount, and other UV absorbing constituents make up the majority of A260. Such samples do not work!

NOTES:

• Use TE to dilute sample, NOT water

• Use small volume pipets (Rainin P2, P10) whenever possible for accuracy

• Best A260 is between 0.1 and 0.6; readings are less accurate outside this range

• A260/A280 ratio of around 2 is a good start

• A spectrum from 210 or 220 – 350 nm is highly recommended. [If the investigator does not have access to a scanning spectrophotometer capable of working with small samples, contact the Center for Medical Genomics for assistance. We can help with this scanning for a small additional charge.]

• If your total RNA samples do not meet the criteria shown in the image (following page), contact CMG for consultation.

• The nucleotide peak should be at 260 nm. A peak at 270 indicates severe contamination and an un-usable sample. The real amount of RNA in such samples is usually only 10-15% of the nominal amount calculated from A₂₆₀. RNA must be repurified before use!

• The leading 210 nm peak should be lower than the 260 nm peak, with a distinct trough at about 230 nm. Ratio of 260 to 230 should be about 2.

• If the total RNA does not meet both of these spectra standards, re-purify your samples through Qiagen RNeasy.