TITLE: RNA Extraction with Trizol
AUTHORS: Dakota Betz

[DESCRIPTION]
This is a protocol for RNA extractions (using Trizol). A maximum of 3 samples can be extracted at once using this protocol. The entire protocol should be run in the fumehood. This protocol is time- and temperature-sensitive, so read all instructions before attempting and make sure you are adequately prepared.

[BEFORE_START]
Celean all surfaces, tools, and gloves with RNase Away. Don't forget to clean pipettes, plastic pipettes, ice blocks, centrifuge, etc. 
Make sure buffers have been prepped if it's a new kit (instructions in kit for which reagents/how much to add to each).

[GUIDELINES]
A maximum of 3 samples can be extracted at once using this protocol.

[STEPS]
SECTION: Homogenize Tissues
1. Add 770 uL of Trizol to bead bashing tube (1 per sample).

SECTION: Homogenize Tissues
2. Pour tissue into a clean Petri dish, keep on cooling block (flat ice pack).
SECTION: Homogenize Tissues
3. Transfer pieces of tissue to bead bashing tube, blot RNAlater with a kim tissue. 
May be a very small amount of tissue (if so use all) and blot until it’s REALLY dry. Do all of this on top of an ice pack to keep it cold.
SECTION: Homogenize Tissues
4. Beadbash 3-4 times for 15 sec at max speed. Cool down 5-10 seconds in between, placing tube in/on ice block. Stop as soon as tissue is dissolved!
SECTION: Homogenize Tissues
5. Transfer liquid to a 1.6 mL RNase free tube and centrifuge 12000 x g, 2 min. Use block from lab freezer to keep tubes cold.

SECTION: Homogenize Tissues
6. Transfer supernatant to a new 1.6 mL RNase free tube.
SECTION: RNA purification &amp; DNase I treatment
7. Add 700 uL of 100% ethanol to each tube. Vortex.

SECTION: RNA purification &amp; DNase I treatment
9. 1) Discard flow-through. Repeat with remaining 700 uL (if applicable). If first half did not flow through completely, centrifuge12000 x g, 2 min .

SECTION: RNA purification &amp; DNase I treatment
10. 2) Transfer column to  RNase-free tube and do in-column digestion. Wash with 400 uL of RNA Wash Buffer. Centrifuge 12000 x g, 30 s, discard flow-through.

SECTION: RNA purification &amp; DNase I treatment
11. 3) Add 80 uL of DNase I Reaction Mix (75 µL (kit), 5 µL (freezer, green cap); scaled) directly to the column. Incubate for  15 min. Centrifuge  12000 x g, 30 s.

SECTION: RNA purification &amp; DNase I treatment
12. 4) Add 400 uL of Direct-zol RNA PreWash. Centrifuge 12000 x g, 1 min.

SECTION: RNA purification &amp; DNase I treatment
13. 5) Discard flow through. Repeat previous step. (Pre-wash and centrifuge again).
SECTION: RNA purification &amp; DNase I treatment
14. 6) Add 700 uL of RNA Wash Buffer. Centrifuge 12000 x g, 1 min. Discard flow-through. Centrifuge again in emptied collection tube 12000 x g, 2 min.

SECTION: RNA purification &amp; DNase I treatment
15. 7) Transfer column to labeled RNase free tube.
SECTION: RNA purification &amp; DNase I treatment
16. 8) Add 26 uL of RNase-free water to column. Incubate 3 min. Centrifuge 12000 x g, 1 min.

SECTION: RNA purification &amp; DNase I treatment
17. 9) Combine tubes if applicable (see note on step 8). Take 5 uL for QC/Qubit. The kit is called “Qubit RNA HS” and has weirdly clear dye and different standards.

SECTION: RNA purification &amp; DNase I treatment
18. 10) Store everything at -80 °C

SECTION: RNA purification &amp; DNase I treatment
8. Load 700 uL into ZymoSpin IIC Column in the 2mL-size RNase-free collection tube. Use this size for all remaining steps. Centrifuge 12000 x g, 1 min.