Is Your RNA Concentration Measurement Accurate? Tips for Better Results

In summary, the recommended protocol for measuring the concentration of RNA is to use UV spectrometry with a dilution factor of 1000/10 and measure absorbance at 260 nm, 280 nm, and 320 nm. The RNA concentration can be calculated using the formula (Net A260 - Net A320) x 40 (extinction coefficient for RNA). The purity of the sample can also be determined by calculating the ratio of Net A260 to Net A280, with a target range of 1.7 to 2.0 for pure RNA. It is important to ensure that the RNA is well mixed in the diluent and that the spectrophotometer is functioning properly. If there are any issues with the
  • #1
mountain
53
0
Do you have any good protocols for measuring the concentration of RNA? The ones i have they seem to be very confused.


Thanks.
 
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  • #2
I usually used UV spectrometry. I put 10 uL of RNA into 990 uL of RNase free water. My blank is 5 uL of the suspension liquid for your RNA into 990 uL of RNase free water.

You measure the Absorbance at 260 nm, 280 nm and 320 nm. 260 is for nucleic acid, 280 is for protein and 320 is your background.

(Net A260 - Net A320 ) x RNA extinction coefficient (40 for RNA) x dilution factor (1000/10) = RNA concentration in ug/mL

Purity of sample = Net A260/Net A280. for pure RNA you should have a ratio of 1.7 to 2.0

If you have problem with the reading, it might be due to the fact that your RNA is not well mixed in the dilutent. You can heat up the sample at 55C for 10 minutes and then take the reading. Also make sure your DNA is well resupended in the sample you will used for measuring the concentration. Vortex the sample for about 5 sec prior to taking the reading.

Also used the same cuvette for your blank and sample. Make sure the cuvette is cleaned between sample. You should rince with water about 20 times.
 
  • #3
iansmith said:
If you have problem with the reading, it might be due to the fact that your RNA is not well mixed in the dilutent. You can heat up the sample at 55C for 10 minutes and then take the reading. Also make sure your DNA is well resupended in the sample you will used for measuring the concentration. Vortex the sample for about 5 sec prior to taking the reading.

If you have problems with the readings, sometimes it's also worth trying another spectrophotometer! I was going nuts once trying to figure out why I was having such a problem with purity/yield of my samples, and then someone suggested I try the spectrophotometer in another lab. Sure enough, there was a problem with the equipment, not my samples. :grumpy:
 

1. How is RNA concentration measured?

RNA concentration can be measured using a variety of methods, such as spectrophotometry, fluorometry, and quantitative PCR (qPCR). These methods involve measuring the absorbance, fluorescence, or amplification of RNA molecules in a sample.

2. Why is measuring RNA concentration important?

Measuring RNA concentration is important because it allows scientists to determine the amount of RNA present in a sample, which can be used to assess the quality and quantity of RNA for downstream experiments. It is also useful for comparing RNA levels between different samples or experimental conditions.

3. What are the units of measurement for RNA concentration?

The most common units of measurement for RNA concentration are nanograms per microliter (ng/μL) or micrograms per milliliter (μg/mL). However, other units such as moles per liter (mol/L) or copies per microliter (copies/μL) may also be used.

4. How is RNA concentration affected by factors such as sample storage and handling?

RNA concentration can be affected by various factors, such as the type of sample, storage temperature, and exposure to RNases. It is important to follow proper sample handling and storage protocols to minimize any potential impact on RNA concentration.

5. Are there any limitations to measuring RNA concentration?

While measuring RNA concentration is a useful technique, it does have limitations. For example, some methods may be more sensitive than others and may only detect a limited range of RNA concentrations. Additionally, certain contaminants or impurities in a sample may interfere with accurate measurements. It is important to choose the appropriate method and consider any potential limitations when measuring RNA concentration.

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