RNA Purification from Collagen Gels (Qiagen)

Youssef Farhat, Written 5/9/11, Last Updated 5/2/13

Introduction

Extracting RNA from water-rich samples such as collagen gels can be challenging at first.  It took me a few months to establish a protocol that yielded sufficient amounts of RNA from my gels with high purity and consistency.  I used this protocol most extensively for gels that were ~400 microliters in volume and contained about 300,000 cells.  While this protocol calls for the more commonly used Qiagen’s RNeasy kit (below), a quick price comparison will reveal that the Epoch Life Science’s Kit is a much cheaper alternative that performs just as well (see related page).

Qiagen’s RNeasy Kit

Materials

  • Trizol (Invitrogen, Cat. #15596-026)
  • Chloroform (Sigma-Aldrich, Cat. #C2432-25ML)
  • 70% EtOH (RNase-free)
  • Qiagen’s RNeasy mini Kit (Cat. #74104)
  • RNase-free Eppendorf tubes
  • RNase free water (Gibco, Cat. #10977)

Procedure

  1. Freeze gels in 1.5 mL Eppendorf tubes at -80ºC until ready for RNA extraction
  2. Add 1 mL Trizol (20ºC) to the 1.5 mL Eppendorf tubes containing each sample
  3. Vortex the tubes for 10-20 seconds until the gels float in Trizol and are fully immersed
  4. Homogenize the gels with 20-G needles and 5 mL syringes until they are totally dissolved
  5. Allow the Trizol-gel mixtures to sit for 5 min at 20ºC
  6. Add 200 μL chloroform to each tube, shake them vigorously for 15 sec, and then wait 2 min at 20ºC for the phases to separate
  7. Centrifuge the tubes at 12,000 g for 15 min (5ºC) and, while waiting, pre-label 2 new RNase-free Eppendorf tubes and one RNeasy column for every sample
  8. Fill one of the newly labeled Eppendorf tubes with 500 uL of 70% EtOH
  9. Transfer the upper aqueous phase of each sample to the appropriate EtOH-filled Eppendorf tube and mix this phase thoroughly with the EtOH by pipetting several times (Note: when removing the upper aqueous phase, be careful not to disturb the other layers of the Trizol separation, as this will contaminate the RNA)
  10. Add 500 uL of the EtOH-RNA solution to the pre-labeled RNeasy Mini Spin columns
  11. Centrifuge at 8000 g for 15-30 sec (5ºC) & carefully empty the flow-through so as not to let any liquid touch the bottom of the RNeasy column (to prevent contamination of the RNA)
  12. Repeat steps 10 and 11 with the remaining EtOH-RNA
  13. Add 700 μL Buffer RW1 to each column, centrifuge at 8000 g for 15 sec, & empty flow-through
  14. Add 500 μL Buffer RPE to each column, centrifuge at 8000 g for 15 sec, & empty flow-through
  15. Add 500 μL Buffer RPE to each column, centrifuge at 8000 g for 2 min, & empty flow-through, discard the collection tube
  16. Place column in a new collection tube and centrifuge at 8000 g for 1 min to eliminate any remaining wash solution from the columns
  17. Place column in the second 1.5 mL Eppendorf tube that was labeled in step 7
  18. Add 34 μL RNase-free water directly to membrane and let set 1 min
  19. Centrifuge at 8000 g for 2 min
  20. The eluted water should contain RNA which is ready to be measured and reverse-transcribed.  See our post on Measuring RNA with NanoDrop for more information.
  21. When finished, store remaining RNA at -80ºC

Helpful References

Citing this Page

Farhat, Y. “RNA Extraction from Collagen Gels Using Qiagen’s RNeasy Kit.” The Protocol Place. March 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com>

2 Responses to RNA Purification from Collagen Gels (Qiagen)

  1. Thanks for the protocol- what do your 260/230 ratios look like and what is your average yield? I’ve been following a similar protocol and am disappointed in my RNA yield. Just wondering how mine compares to yours! Thanks again.

    • Thanks for the comment. I have addressed your questions in the page called “Measuring RNA with NanoDrop” – see http://protocol-place.com/basic-lab-techniques/rna-protocols/measuring-rna-yield-with-a-nanodrop-spectrophotometer/ . I just updated this protocol so that it links to that page now (Step 20).

      You didn’t mention how many cells are in your samples, but I’ve noticed that samples with fewer cells yield less RNA and also lower A260/230 ratios. Try increasing cell numbers if you think that having too few cells might be an issue. It could be that your cells are dying too, causing less yield and lower quality. For example, if you are making cell-seeded collagen gels, be sure the pH of the collagen is neutral each time and if you can check any other way, be sure that your cells are actually surviving the experiment. See below for more info, and good luck!

      Copied from the other page mentioned above:

      “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.”

      “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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