RNA and protein extraction from bulk dissections

(1st time): Add 375ul of 2-mercaptoethanol to 2X Denaturing Solution , Add 21ml of 100% Etanol to miRNA Wash Solution 1 , Add 40ml of Etanol 100% to Wash Solution 2/3 .

Preheat Nuclease-free Water (#129114, Qiagen-Werfen) at 95oC (for elution).

Preheat 2x Denaturing Solution to 37^oC until it is completely in solution (to dissolve crystals formed when stored at 4^oC).

Keep bulk dissections in dry ice until adding to cell disruption buffer.

Add Protease/phosphatase inhibitor cocktail 100x (1/100) (#5872S, Werfen - Cell signaling) to Cell disruption buffer .

Add 500ul of Cell disruption buffer into 2mL eppendorfs. This volume can range from 100 to 625ul as stated in the protocol (~50mg tissue-->500 ul to each sample)* and keep the buffer on ice.

Add the frozen bulk dissections to the 2mL eppendorfs with Cell disruption buffer.

Disrupt the samples using a Omni Tissue Homogenizer (TH) . Disrupt for no more than 5s and then put it back on ice, to keep the sample cold during homogenization. If you still see clumps of tissue homogenize 5s more. Try not to take the rotor out of the cell disruption buffer to avoid creating foam.

Split the sample for RNA and PROTEIN: 200ul RNA and 200ul Protein (approximately 100ul is lost as foam). Leave protein sample on ice for a minimum of 10 min or more, and proceed with the RNA sample.

Add Denaturing Solution 2x . Same volume as the sample split for RNA extraction (-->200ul). Denaturing buffer contains β –mercapto so it has to be manipulate din an appropriate fume-hood.

10. Incubate 5min on ice.

11. Add Acid-Phenol: Chloroform . Same volume as the actual sample (volume sample split for RNA extraction + volume 2x Denaturing Solution (-->400 ul) (Take the bottom phase of the bottle of Acid-Phenol:Chloroform , not the aqueous phase on top).

12. Vortex 30-60s.

13. Centrifuge 8min at max. speed. If interphase is not compact centrifuge again.

14. Remove the upper aqueous phase (avoiding interphase) and transfer to a new tube**.

15. Add 1,25x volumes of Ethanol 100% (at room temperature).

16. Pipette up and down 3 times and transfer to filter column (max. 700ul, if not perform successive transfers).

17. Centrifuge 30s at 10.000g. Discard flow-through.

18. Add 700ul miRNA Wash Solution 1 . Centrifuge 15s at 10.000g. Discard flow-through.

19. Add 500 ul de Wash Solution 2/3 . Centrifuge 15s at 10.000g. Discard flow-through(X2).

20. With the same tubes centrifuge 1min at 10.000g to eliminate residual fluid.

21. Transfer the column to a new Collection tube (provided). Add 30ul Nuclease-free water pre-warmed at 95oC to elute RNA.

22. Centrifuge 30s at 10.000g. Collect eluate.

23. Re-load the eluate in the column, centrifuge again 30s at 10.000g. Quickly place the samples on ice (RNA is now in water solution and is highly labile).

24. (Optional) Take 1,5ul from the extracted RNA sample for Bioanalyzer analysis.

25. (Optional) Take 1,5ul for RNA concentration quantification with NanoDrop ND-1000 Spectrophotometer.

26. As fast as possible store the samples at -80oC.

27. Take the split sample for protein extraction and centrifuge at 4oC at 10.000g for 2min.

28. Collect the supernatant and transfer it to a new tube.

29. Sonicate 3x5min (30s on ice between intervals).

30. Store at -80oC.