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)

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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

• Untergasser Lab Protocol
• Qiagen’s RNeasy Kit

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>