Mar 06, 2023
Sigma GeneElute total RNA extraction from fungal tissue
- 1Natural History Museum of Utah
Protocol Citation: ALEXANDER J BRADSHAW 2023. Sigma GeneElute total RNA extraction from fungal tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7znyv5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 24, 2022
Last Modified: March 07, 2023
Protocol Integer ID: 57351
Abstract
RNA extraction from fungal tissue using the Sigma GeneElute total RNA kit.
Tissue and workspace preparation
Tissue and workspace preparation
15m
15m
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
Flash Freeze tissue in Liquid Nitrogen (LN2).
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.
At this point frozen ground tissue can be stored at -80C for later extraction.
RNA extraction
RNA extraction
15m
15m
add 600ul of buffer RL to a 1.7ml eppendorf tube.
Place no more than 50mg of ground tissue into the sample tube with buffer RL.
Incubate for 15-30 minutes at room temperatureRoom temperature
15m
Centrifuge at maximum speed for 2 minutes
Equipment
Centrifuge
NAME
Benchtop Centrifuge
TYPE
Eppendorf
BRAND
5405000441
SKU
LINK
Any benchtop centrifuge will suffice
SPECIFICATIONS
Transfer the lysate to a new RNAse free 1.7ml tube.
Add 1:1 volume (so 600ul) of freshly prepared 70% etoh
Vortex for 10 seconds, or until homogenous
Assemble a column with a waste tube from the GeneElute kit.
Add 600ul of lysate/etoh solution to the assembled column
Centrifuge at 6000G for 2 minuts
If lysate is not completely passes, spin at an additional 14,000G for 1 minute
Discard waste from waste tube, and repeat step 13 and 14 if more lysate/etoh exists.
Place column into a new waste collection tube
Add 400ul of wash solution A to column
Centrifuges at max speed for 1.5 minutes
discard waste from collection tube
Repeat steps 17-19 two more times (for a total of 3x wash steps)
Centrifuge Sample for a final 2 minute dry spin at max speed
Place column in a clean eppendorf tube provided in the GeneElute kit.
add 50ul of elution solution A
Centrifuge at 200G for 2 minutes
centrifuge at 14,000G for 1 minute
If the entire 50ul has not eluted, then spin for an additional minute at 14,000G
Quality control of RNA and storage
Quality control of RNA and storage
15m
15m
Keep eluted samples on Ice, Prepare a bleach gel for RNA analysis
BLEACH GEL:
100ml TAE
400ul concentrated bleach
1g agarose
Make sure to add bleach before microwaving.
Load and run samples normally at 120V for ~20 minutes with a 100bp ladder.
immediately store samples at -80C after loading gel.