TITLE: Sigma GeneElute total RNA extraction from fungal tissue
AUTHORS: Alexander  J Bradshaw

[DESCRIPTION]
RNA extraction from fungal tissue using the Sigma GeneElute total RNA kit. 

[STEPS]
SECTION: Tissue and workspace preparation
2. Flash Freeze tissue in Liquid Nitrogen (LN2).
SECTION: Tissue and workspace preparation
3. Grind Fungal Tissue to a fine powder with a mortar and pestle with LN2, be careful to not allow the tissue to thaw during this Process. 
SECTION: Tissue and workspace preparation
3.1. At this point frozen ground tissue can be stored at -80C for later extraction.
SECTION: RNA extraction
4. add 600ul of buffer RL to a 1.7ml eppendorf tube. 
SECTION: RNA extraction
5. Place no more than 50mg of ground tissue into the sample tube with buffer RL.
SECTION: RNA extraction
6. Incubate for 15-30 minutes at room temperatureRoom temperature 

SECTION: RNA extraction
7. Centrifuge at maximum speed for 2 minutes
 

SECTION: RNA extraction
8. Transfer the lysate to a new RNAse free  1.7ml tube. 
SECTION: RNA extraction
9. Add 1:1 volume (so 600ul) of freshly prepared 70% etoh
SECTION: RNA extraction
10. Vortex for 10 seconds, or until homogenous 
SECTION: RNA extraction
11. Assemble a column with a waste tube from the GeneElute kit. 
SECTION: Tissue and workspace preparation
1. ALL WORK MUST BE DONE IN THE STERILE HOOD THAT HAS BEEN CLEANED FOR RNA WORK

REGULARLY CHNAGE GLOVES AND  WASH HANDS WITH RNASE TO AVOID DEGREDATION

MAKE SURE ALL PIPETORS, TIPS, AND ANY OTHER EQUIPMENT ARE IN THE HOOD AND CLEANED WITH RNASE

DO NOT MOVE ANYTHING IN OR OUT WITHOUT WASHING WITH RNASE

Reagents that need to be made:
Fresh, day of 70% etoh
SECTION: RNA extraction
12. Add 600ul of lysate/etoh solution to the assembled column
SECTION: RNA extraction
13. Centrifuge at 6000G for 2 minuts
SECTION: RNA extraction
14. If lysate is not completely passes, spin at an additional 14,000G for 1 minute
SECTION: RNA extraction
15. Discard waste from waste tube, and repeat step 13 and 14 if more  lysate/etoh exists. 
SECTION: RNA extraction
16. Place column into a new waste collection tube
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17. Add 400ul of wash solution A to column
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18. Centrifuges at max speed for 1.5 minutes
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19. discard waste from collection tube
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20. Repeat steps 17-19 two more times (for a total of 3x wash steps)
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21. Centrifuge Sample for a final 2 minute dry spin at max speed
SECTION: RNA extraction
22. Place column in a clean eppendorf tube provided in the GeneElute kit. 
SECTION: RNA extraction
23. add 50ul of elution solution A
SECTION: RNA extraction
24. Centrifuge at 200G for 2 minutes
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25. centrifuge at 14,000G for 1 minute
SECTION: RNA extraction
26. If the entire 50ul has not eluted, then spin for an additional minute at 14,000G
SECTION: Quality control of RNA and storage
27. Keep eluted samples on Ice, Prepare a bleach gel for RNA analysis
SECTION: Quality control of RNA and storage
28. BLEACH GEL:
100ml TAE
400ul concentrated bleach
1g agarose

Make sure to add bleach before microwaving. 
SECTION: Quality control of RNA and storage
29. Load and run samples normally at 120V for ~20 minutes with a 100bp ladder. 
SECTION: Quality control of RNA and storage
30. immediately store samples at -80C after loading gel.