RNA extraction from cells in 3D culture IPNs or collagen gels

Calcium chelation and sample homogenization:

1. Set microcentrifuge to 4°C and prepare ice bucket.

2. Place PBS + 50 mM EDTA on ice.

3. On ice, grind each tube containing 2 frozen 500 µl gels into dust (or as close as possible).

4. Add 500 µl ice cold PBS + 50 mM EDTA to each sample. Incubate on ice for 5 min, pipetting to break up stiff IPNs (collagen gels will largely remain intact).

5. In hood, on ice, add 2 ml Trizol to each tube and homogenize using 20 G needle.

6. Aliquot each sample into 2 - 1.5 ml eppendorf tubes. Spin at 12,000 x g for 10 min at 4°C to pellet alginate.

7. Transfer cleared supernatant into 2 new eppendorf tubes, ~1.2 ml per tube.

Phase separation:

1. Incubate sample for 5 min at RT to permit dissociation of nucleoprotein complex.

2. In hood, add 200 µl chloroform to each tube.

3. Vortex vigorously for 15 s.

4. Incubate for 3 min at RT.

5. Centrifuge at 12,000 x g for 15 min at 4°C.

RNA precipitation:

1. Following centrifugation, sample should have separated into 3 phases:

1. top: clear aqueous (~50%) - contains RNA

2. middle: interphase

3. bottom: red phenol-chloroform

2. Carefully transfer clear aqueous phase to new tube using 200 µl pipette tips. Leave ~100 µl of aqueous phase in old tube to be sure NOT to contaminate with interphase. Should have about 400-600 µl aqueous phase in new tube, discard rest of solution in Trizol/chloroform waste.

3. Add 500 µl 100% ethanol to each tube. Mix well by inverting tubes several times.

4. Transfer 700 µl of sample, including any precipitate, into spin column placed in collection tube. Both tubes from above are combined onto 1 spin column.

5. Centrifuge for 30 s at 10,000 x g. Discard flow through.

6. Transfer remaining volume of sample onto the spin column. Repeat spin and discard flow through.

7. Wash the column by adding 500 µl WF Buffer. Centrifuge for 30 s at 10,000 x g. Discard flow through.

8. Wash the column by adding 700 µl WS Buffer. Centrifuge for 30 s at 10,000 x g. Discard flow through.

9. Centrifuge for 3 min at 10,000 x g to remove residual ethanol.

10. Place column into new 1.5 ml eppendorf tube. Add 30 µl RNase free ddH20 onto center of membrane.

11. Incubate at RT for 1 min.

12. Centrifuge for 1 min at 10,000 x g.

13. Quantify RNA concentration using NanoDrop. Yield should be ~0.5-1 µg RNA (for 1 ml of gel at 105 cells/ml and 7 days in culture). Store RNA at -80°C.

Modified from Fan Yang Lab and RNeasy Mini Kit Protocol