RNA quantification and quality control 

You now have two samples (WT and mutant) of RNA purified from your samples. Before carrying out the reverse transcription, which is usually the most expensive part of the protocol, we will quantify and test the quality of the RNAs. 

A. Quantifying the RNA will allow us to know the volume to use to get 1 µg of RNA in order to perform the reverse-transcription step. This amount should be the same for the two samples and can be adapted if needed (e.g. if you have lower yields). 

B. Assessing the quality of the RNA is necessary to make sure we use an intact, non-contaminated mRNA samples for the RT step.

Material

  • RNA samples
  • 1,5 ml Eppendorf
  • Sterile water
  • A spectrophometer for nucleic acid dosing, such as a Nanodrop or a BioSpec-nano machine (we will use the first one: https://tinyurl.com/ykkyhh7f)

Procedure

  1. Prepare a 1,5 ml Eppendorf with 50 µl water (will be used to normalize the measurements)
  2. Contact us to mention that you are ready for RNA quantification.
  3. The BioSpec-nano will be operated by a supervisor :
    • Add 2 µl of H2O to the BioSpec and make the calibration ;
    • Add 2 µl of H2O to the BioSpec to check that the calibration is correct;
    • Add 2 µl of samples and make the measurements. Write down the results and repeat the measurements for each sample. If the second measurement differs from the first by > 10%, repeat the measurement a third time.

This step will provide you with three types of information :

A. The RNA concentration The concentration is usually measured in ng/µl. Typical yields range from 50 to 1000 ng/µl, depending on the quantity and origin of the tissues (plant species, tissues, age of the plants, etc). Usually, the reverse transcription is performed on a total amount of 750 to 1500 ng of RNA. Since the RNA volume used for the RT is usually of maximum 10 µl, you need to have ≥ 150 ng/µl to carry out the reverse transcription of 1500 ng of RNA. If your sample yields are lower, the total amount of RNA used for the RT will be adapted. However, for a given experiment, all samples will use the same amount of RNA, so that the sample with the lower yield will define the total quantity used for the RT in all samples.

B. The 260/280 Ratio The ratio between the absorbance at 260 and 280 nm is an indicator of RNA purity. A ratio ~ 2.0 indicates a pure sample (in this case, a ratio >1.8 is still acceptable). Lower ratios indicate the presence of contaminants such as proteins, phenols or any other contaminant that has an absorbance close to 280 nm.

C. The 260/230 Ratio That ratio is used as a secondary measurement of nucleic acid purity. The 260/230 values for “pure” RNA samples usually range around 2-2.2. A ratio of 1.8 is still acceptable but lower ratios indicate the possible presence of contaminants that absorb at 230 nm (e.g. EDTA, phenol, carbohydrates, TRIzol).

Hopefully, you will obtain good quality RNAs. In case you do not have RNA at all, you will use samples from other groups. In case RNAs are of poor quality, the decision to continue with these samples or to use RNAs from other groups will be taken depending on the ratios you obtained.

Here is an example (found here) of the alteration of the OD curve observed for contaminated versus non-contaminated nucleic acids (DNA, in this case):

Example image