TITLE: RNA Isolation of Human Meniscus
AUTHORS: Takuya Sakamoto, Merissa Olmer, Martin Lotz

[DESCRIPTION]
This protocol demonstrates how to perform RNA isolation from fresh snapfrozen and AllProtect preserved human meniscus tissue.

[BEFORE_START]
This protocol works with both fresh snapfrozen and AllProtect preserved tissue.

[STEPS]
SECTION: Tissue Homogenization
2. Precool Benchtop Centrifuge and make sure temperature is at 4℃.
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3. Prepare tools, mortar and pestle, and work surface by spraying down with RNAse Away.
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4. Weigh out 100mg of preserved tissue. If you have to cut a smaller piece of tissue to get 100mg, do so in a petri dish placed on a piece of dry ice so sample remains frozen.
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5. Add two ladles of liquid nitrogen to a clean mortar and pestle; allow to become cold.
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6. After about half of liquid nitrogen has evaporated, add snapfrozen or AllProtect tissue to the mortar.
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7. Add another ladle of liquid nitrogen to mortar and allow about half of the liquid nitrogen to boil off. 
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8. Begin smashing and grinding up tissue with the pestle; this is an ongoing process so keep adding liquid nitrogen until grinding creates a fine powder. Do not let the mortar and sample get warm.
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8.1. Note - samples have a tendency to pop out of the mortar, having a silicone lid top on the mortar in the beginning helps keep tissue contained.
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9. Once you have the desired consistency of a fine powder, add half a ladle of liquid nitrogen and tilt mortar slightly on its side and begin scraping liquid nitrogen/powdered tissue suspension to the bottom for a more thorough collection.
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10. Just as the liquid nitrogen evaporates, use a pre-chilled scoop to transfer tissue powder right away into a chilled CK28R tube with Trizol.
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11. Place cooled CK28R tubes with samples in the Precellys and use 6500rpm, 20 second cycles for a total of 4 times. Cool tubes on ice for 3 minutes between each cycle.  
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12. Once samples are finished on the Precellys with 4 cycles run, transfer Trizol to a new 1.5mL tube.
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13. You may continue OR stop here and immediately place sample into a -80℃ freezer for next day RNA extraction.
SECTION: RNA Extraction Using Zymo Direct-zol MicroPrep Kit
14. Preparation before starting the isolation protocol:
i. Pre-cool Benchtop Centrifuge to 4℃ AND make sure you have another benchtop centrifuge at Room Temperature.
ii. Prepare Digestion Buffer (DNaseⅠ 5ul x   +1, DNA Digestion Buffer 35ul x   +1) according to Zymo's protocol.
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16. Spin at 12,000g for 15 minutes in 4℃ benchtop centrifuge.
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17. Transfer top aqueous layer (about 400uL) to a Room Temperature 1.5mL microtube.  Be careful to not disturb the middle and bottom layers.
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21. Add 1:1 of 100% Ethanol to the tube with aqueous phase.  (For example-  400uL top layer and 400uL 100% Ethanol).  
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22. Mix Ethanol and aqueous phase gently by pipetting and then add 400uL to Zymo column. When loading column, be careful to not get the rim wet. 
SECTION: RNA Extraction Using Zymo Direct-zol MicroPrep Kit
23. Centrifuge at 14,000 rpm for 1 minute at 4℃. 
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24. Discard flow through after spin, invert collection tube and dab tube on paper towel to remove excess liquid.
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26. Add 400uL of Direct-zol RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 1 minute at 4℃. 
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28. Add 40uL Digestion Buffer (see step 14 ii.) to the column matrix. Incubate at Room Temperature for 15 minutes. 
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29. Add 400uL of Direct-zol RNA PreWash Buffer to the column and centrifuge at 14,000 rpm for 1 minute at 4℃. 
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32. Add 700uL of Direct-zol RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 1 minute at 4℃.  
SECTION: RNA Extraction Using Zymo Direct-zol MicroPrep Kit
33. Discard flow-through and the collection tube. Put the column in a new collection tube. 
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34. Centrifuge for 2 minutes at 14,000 rpm at 4℃.  
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35. Transfer the column carefully into a 1.5mL tube. 
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36. Add 15uL of DNase/RNase-Free Water to the membrane to elute RNA. Centrifuge at 14,000 rpm for 1 minute at 4℃.
SECTION: RNA Extraction Using Zymo Direct-zol MicroPrep Kit
37. Check RNA concentration using Nanodrop. 
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1. Add 1 mL Trizol/TRI Reagent to CK28R bead tubes and place tubes on ice. 
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4.1. If sample was stored in AllProtect, briefly rinse in PBS and wipe off excess liquid with Kimwipes prior to weighing out tissue. 
SECTION: RNA Extraction Using Zymo Direct-zol MicroPrep Kit
15. Add 200uL chloroform (20% volume) to the Trizol and vortex for 30 seconds. If Trizol samples were previously frozen at -80℃, gradually thaw samples on wet ice. 
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18. Add 80uL (20% volume) of chloroform, vortex for 30 seconds. Incubate tubes for 10 minutes at Room Temperature. 
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19. Spin at 12,000g for 15 minutes in 4℃ benchtop centrifuge.
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20. Transfer 400uL of the aqueous phase into 1.5mL microtube. 
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27. Discard flow-through. 
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30. Discard flow-through. 
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31. Repeat Steps 28 and 29.
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25. Repeat Steps 22-23 so that all of the mixture has gone through the column.