RNA Purification from Collagen Gels (Epoch)

Youssef Farhat, Written 9/9/11, Last updated 5/6/14

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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.  The Epoch Life Science’s Kit (below) used in this protocol is a much cheaper alternative to Qiagen’s RNeasy Kit (see related post).  Although it’s cheaper, I found it to yield equally good quality and quantity RNA.

Epoch’s GenCatch Kit

Safety Precautions

Several of the reagents used in this procedure are harmful to health (i.e. TRIzol and Chloroform).  Wear eye, face, and hand protection as well as a lab coat throughout the procedure and be sure to review all safety information that comes with these products before using them.

Materials

  • TRIzol (Invitrogen, Cat. #15596-026)
  • Chloroform (Sigma-Aldrich, Cat. #C2432-25ML)
  • 70% EtOH (RNase-free)
  • Epoch Life Science’s GenCatch Total RNA Miniprep Kit (Cat. #1660050)
  • 3 RNase-free Eppendorf tubes per sample
  • 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 Minispin column for every sample
  8. Fill one of the newly labeled Eppendorf tubes with 500 µL 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 (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 µL of the EtOH-RNA solution to the pre-labeled Minispin 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 column (to prevent contamination of the RNA)
  12. Repeat steps 10 and 11 with the remaining EtOH-RNA
  13. Add 500 μL WF Buffer to each column, centrifuge at 8000 g for 15 sec, & empty flow-through
  14. Add 700 μL WS Buffer to each column, centrifuge at 8000 g for 15 sec, empty flow-through, & discard the collection tube
  15. Place columns in new, unlabeled 1.5 mL Eppendorf tubes and centrifuge at 8000 g for 3 min to eliminate any remaining wash solution from the columns
  16. Place columns in the second set of 1.5 mL Eppendorf tubes that were labeled in step 7
  17. Add 34 μL RNase-free water directly to membrane and let set 1 min at 20ºC
  18. Centrifuge at 8000 g (5ºC) for 2 min
  19. 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.
  20. When finished, store remaining RNA at -80ºC

Helpful References

Citing This Page

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

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