TITLE: RNA and protein extraction from bulk dissections
AUTHORS: miquel.vila

[DESCRIPTION]
mirVana PARIS RNA and Native Protein Purification Kit (#AM1556, Thermo Fisher Scientific )

[GUIDELINES]
*We have less tissue than 0,5mg but 500 ul is a comfortable volume to work with since we have to homogenize the sample with a motorized rotor-stator homogenizer that needs a big volume to homogenize the whole sample. And, afterwards, we have to split the volume into RNA and Protein samples, so 500ul enables a 200 and 200ul separate homogenates (~100ul gets lost as foam) which is a correct volume.
** We suggest collecting only 200ul to avoid collecting interphase. But it is possible to collect 300ul from the aqueous phase if the interphase is compact.

[STEPS]
SECTION: First Time
1. (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.
SECTION: Sample disruption
2. Preheat Nuclease-free Water (#129114, Qiagen-Werfen) at 95oC (for elution).
SECTION: Sample disruption
3. Preheat 2x Denaturing Solution to 37 oC until it is completely in solution (to dissolve crystals formed when stored at 4 oC).
SECTION: Sample disruption
4. Keep bulk dissections in dry ice until adding to cell disruption buffer.
SECTION: Sample disruption
5. Add Protease/phosphatase inhibitor cocktail 100x (1/100) (#5872S, Werfen - Cell signaling) to Cell disruption buffer.
SECTION: Sample disruption
6. 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.
SECTION: Sample disruption
8. 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.
SECTION: Sample disruption
9. 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.
SECTION: RNA extraction
10. 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.
SECTION: RNA extraction
11. 10.   Incubate 5min on ice.
SECTION: RNA extraction
12. 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).
SECTION: RNA extraction
13. 12.   Vortex 30-60s.
SECTION: RNA extraction
14. 13.   Centrifuge 8min at max. speed. If interphase is not compact centrifuge again.
SECTION: RNA extraction
15. 14.   Remove the upper aqueous phase (avoiding interphase) and transfer to a new tube**.
SECTION: RNA extraction
16. 15.   Add 1,25x volumes of Ethanol 100% (at room temperature).
SECTION: RNA extraction
17. 16.   Pipette up and down 3 times and transfer to filter column (max. 700ul, if not perform successive transfers).
SECTION: RNA extraction
18. 17.   Centrifuge 30s at 10.000g. Discard flow-through.
SECTION: RNA extraction
19. 18.   Add 700ul miRNA Wash Solution 1. Centrifuge 15s at 10.000g. Discard flow-through.
SECTION: RNA extraction
20. 19.   Add 500 ul de Wash Solution 2/3. Centrifuge 15s at 10.000g. Discard flow-through(X2).
SECTION: RNA extraction
21. 20.   With the same tubes centrifuge 1min at 10.000g to eliminate residual fluid.
SECTION: RNA extraction
22. 21.   Transfer the column to a new Collection tube (provided). Add 30ul Nuclease-free water pre-warmed at 95oC to elute RNA.
SECTION: RNA extraction
23. 22.   Centrifuge 30s at 10.000g. Collect eluate.
SECTION: RNA extraction
24. 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).
SECTION: RNA extraction
25. 24.   (Optional) Take 1,5ul from the extracted RNA sample for Bioanalyzer analysis.
SECTION: RNA extraction
26. 25.   (Optional) Take 1,5ul for RNA concentration quantification with NanoDrop ND-1000 Spectrophotometer.
SECTION: RNA extraction
27. 26.   As fast as possible store the samples at -80oC.
SECTION: Protein extraction
28. 27.   Take the split sample for protein extraction and centrifuge at 4oC at 10.000g for 2min.
SECTION: Protein extraction
29. 28.   Collect the supernatant and transfer it to a new tube.
SECTION: Protein extraction
30. 29.   Sonicate 3x5min (30s on ice between intervals).
SECTION: Protein extraction
31. 30.   Store at -80oC.   
SECTION: Sample disruption
7. Add the frozen bulk dissections to the 2mL eppendorfs with Cell disruption buffer.