askvity

How to Dilute RNA?

Published in Molecular Biology 3 mins read

To dilute RNA, use RNase-free water (e.g., DEPC-treated water) to achieve your desired final concentration, typically in the range of 0.1-0.5 µg/µl for many applications.

Detailed Steps for RNA Dilution

Here's a step-by-step guide on how to properly dilute your RNA sample:

  1. Prepare RNase-Free Water: Use only RNase-free water (like DEPC-treated water) to avoid RNA degradation by ubiquitous RNases. This is crucial for maintaining the integrity of your RNA.

  2. Determine Initial RNA Concentration: Accurately measure the concentration of your starting RNA sample using a spectrophotometer (e.g., NanoDrop) or fluorometric assay (e.g., Qubit). This is essential for calculating the correct dilution factor.

  3. Calculate Dilution Factor: Use the following formula to calculate the volume of RNase-free water needed:

    • V1 x C1 = V2 x C2

      • Where:
        • V1 = Volume of RNA stock solution needed
        • C1 = Concentration of RNA stock solution
        • V2 = Desired final volume of diluted RNA
        • C2 = Desired final concentration of diluted RNA

    • Rearrange the formula to solve for V1:

      • V1 = (V2 x C2) / C1

  4. Prepare Dilution: Carefully add the calculated volume of RNase-free water to a sterile, RNase-free microcentrifuge tube. Then, add the calculated volume of your RNA stock solution.

  5. Mix Thoroughly: Gently pipette the solution up and down several times to ensure thorough mixing. Avoid vortexing vigorously, as this can shear the RNA.

  6. Aliquot (Optional): If you don't need to use the entire diluted sample immediately, aliquot it into smaller volumes to avoid repeated freeze-thaw cycles, which can degrade RNA.

  7. Store Properly: Store the diluted RNA at -80°C for long-term storage. Avoid storing RNA at -20°C, as this can lead to ice crystal formation and RNA degradation. Flash freezing the RNA by placing it in liquid nitrogen before placing at -80°C can further improve stability.

Example Calculation

Let's say you have an RNA stock solution with a concentration of 1 µg/µl, and you want to dilute it to a final concentration of 0.2 µg/µl in a final volume of 50 µl.

  1. C1 = 1 µg/µl (Concentration of RNA stock)
  2. C2 = 0.2 µg/µl (Desired final concentration)
  3. V2 = 50 µl (Desired final volume)

Using the formula:

V1 = (V2 x C2) / C1

V1 = (50 µl x 0.2 µg/µl) / 1 µg/µl

V1 = 10 µl

Therefore, you should add 10 µl of your RNA stock solution to 40 µl of RNase-free water to achieve a final concentration of 0.2 µg/µl in a total volume of 50 µl.

Important Considerations

  • RNase Contamination: Preventing RNase contamination is paramount. Always use RNase-free consumables (tubes, pipette tips) and work in a clean environment. Wear gloves and change them frequently.

  • Accurate Pipetting: Accurate pipetting is crucial for precise dilutions. Use calibrated pipettes and appropriate techniques.

  • Verification: After dilution, consider verifying the RNA concentration using a spectrophotometer or fluorometric assay to ensure the dilution was performed correctly.

Related Articles