Youssef Farhat, Written 9/9/11, Last updated on 5/2/13
Measuring the amount and purity of purified RNA is crucial for determining the amount of each sample to use in downstream applications, such as reverse transcription or RT-PCR. NanoDrop Spectrophotometers (NDS), such as the one below, are very convenient instruments for assessing RNA quantity and quality.
This is how to use the NDS to measure RNA quantity, followed by a few points on interpreting the 260/280 and 260/230 ratios, important indicators of RNA quality.
- Bring RNA samples as well as the water used to elute them on ice to the spectrophotometer
- Wash the sample reader with molecular grade water and dry with a KimWipe
- Following the software’s instructions, load 2 µL of elution water (blank) and initialize the system
- Change the computer’s setting to RNA and click the blank button
- Load 2 µL of sample and click the measure button
- After the read is complete, record the A260/A280 and A260/A230 ratios as well as the amount of RNA recovered (in ng/µL)
- Wipe the sample reader with a clean, dry KimWipe between samples and repeat steps 5-6.
Interpreting the Results & Troubleshooting
- As a reference, collagen gels seeded with ~700,000 cells typically yield around 50-150 ng/µL of RNA when eluted in 30-35 µL of water
- A lower than expected concentration of RNA indicates low cell numbers in the sample, poor homogenization of samples, or too much volume of water used in the elution step of RNA purification
- Very pure RNA will have an A260/A280 ratio of ~2.1. Anything higher than 1.8 is considered to be of acceptable purity, and a ratio of <1.8 indicates potential DNA or protein contamination. A low A260/A280 ratio is likely due to mixing phases when removing the upper aqueous phase of the Trizol separation or is also more common in samples with a very low yield of RNA.
- The A260/A230 ratio should also be above 2.0. A low A260/230 ratio indicates contamination with the wash solutions, chaotropic salts, phenols or protein. A low A260/A230 ratio is most likely due to contamination of the samples with washing buffers during the Minispin tube washes. Be more careful when handling the tubes, especially when adding wash solution or removing the spin-through. Try to gently pour out the flow-through and then carefully wipe away drops on the outer rim of the collection tube with a KimWipe.
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