TITLE: Modified RNeasy Mini Kit protocol for filter extractions
AUTHORS: Margaret M. Brisbin

[DESCRIPTION]
RNA extraction protocol for filters - modification of RNeasy mini kit protocol

[STEPS]
SECTION: Prep
1. UV sterilize hood and clean all surfaces with RNAse-away
SECTION: Prep
4. Take out and label tubes: 
 
Each sample needs: 
    1x bead-bashing tube
    2x Lo-bind Eppendorf tubes
    1x RNeasy filter column
    1x RNeasy capped collection tube  

number tubes consecutively from last extraction (if last extraction was #126, start numbering at 127)
SECTION: Cell Lysis
7. Transfer filter tubes for that day's extraction into the liquid nitrogen dewar or into a cooler with dry ice
SECTION: Prep
5. Make fresh 70% ethanol with molecular grade ethanol and nuclease-free water 
(need up to 600 µl per sample)

can reuse the 50m falcon tube in the RNA hood
SECTION: Prep
6. Make RLT+BME lysis buffer for the number of samples you have +1 in a falcon tube: 

Per sample / extra: 
    590 µl of RLT buffer 
    10 µl of BME

vortex to mix 

add 600 µl of RLT+BME to each bead-bashing tube 

SECTION: Prep
2. Preheat thermal block to 65ºC
SECTION: Prep
3. Make sure there are enough DNase aliquots for the day's extractions. Move aliquots you'll need from the freezer to the fridge so they are thawed when you need them. 
SECTION: Cell Lysis
8. Using ethanol-cleaned forceps, remove filter half from cryovial and transfer to a bead tube with RLT+BME buffer.

Get as much of the filter as possible, but do not worry if some small fragments get away. 

Push the filter pieces down into the beads and buffer so that the filter breaks apart a bit and all surfaces are covered in buffer. 

Write down all information on the cryovial in lab notebook with the corresponding extraction number   

continue for all filters extracting from that day (max 24, recommend 10 or 12 at the beginning -- 10 is good so all samples can go one bioanalyzer chip )


SECTION: Cell Lysis
9. Transfer all bead tubes to the thermal block mixer and incubate at 65ºC for 10 mins 65 °C 10 min 

SECTION: Cell Lysis
10. attach the bead tube adapter to the vortex-genie 

move all bead tubes to the vortex adapter  (caps facing in) so that the adapter is balanced

Vortex at the highest setting for 5 min 5 min 

SECTION: Cell Lysis
11. Centrifuge bead tubes for 1 min at 4,000 rcf 4000 rcf, 1 min 

SECTION: Cell Lysis
12. transfer bead tubes to RNA hood. Using p1000 pipettor set to 1000 µl remove ALL supernatant from the bead tube and transfer to an eppendorf lo-bind tube. Start by removing liquid above the filter and beads, then "wring-out" thefilter by pressing it against the tube wall and collecting additional supernatant. Finally, push the pipette tip down into the beads to collect as much remaining supernatant as possible. 

It is OK to collect beads and other detritus.  
SECTION: Cell Lysis
13. Centrifuge eppendorf tubes with supernatant from bead tubes for 1 min at 4,000 rcf 4000 rcf, 1 min 

SECTION: Cell Lysis
14. Move tubes back into the RNAhood. Transfer 500-600 µl of supernatant to clean eppendorf lo-bind tubes.  It is OK to have different volumes from different samples, but it makes it much easier to pick the minimum volume you are able to recover and transfer the same volume for all the samples. 
SECTION: Cell Lysis
15. Add an equal volume (500-600 µl) of freshly-prepared 70% EtOH to each sample. 
SECTION: RNA purification
16. transfer up to 700 µl of cell lysate + 70% EtOH to top of spin column. 
SECTION: RNA purification
17. Transfer spin columns to centrifuge and spin at 10,000 rcf for 20s 10000 rcf, 20 s 

SECTION: RNA purification
18. discard flow through. Transfer remaining sample to top of spin column and spin again for 10,000 rcf for 20 s 10000 rcf, 20 s 

SECTION: RNA purification
19. If looking at Qiagen protocol cards, switch over to Part 2 for on-column DNase digestion. 
SECTION: RNA purification
20. Add 350 µl of RW1 to the top of each column. Spin at 10000 rcf for 20 s. Discard flow-through.10000 rcf, 20 s 

SECTION: RNA purification
21. Prepare DNase mastermix for each sample + 1 extra:

10 µl of DNase stock 
70 µl of RDD Buffer (in DNase kits on door of older fridge) 
SECTION: RNA purification
22. Add 80 µl of DNase master mix directly to the center of each spin column. Incubate for 15 minutes at room temperature. 15 min 

SECTION: RNA purification
23. Add 350 µl of RW1 to the top of each spin column. Centrifuge at 10000 rcf for 20s and discard flow-through. 10000 rcf, 20 s 

SECTION: RNA purification
24. If looking at Qiagen Protocol, go back to part one and start at step 5. 

Add 500 µl of RPE to spin column. Centrifuge at 10000 rcf for 20s and discard flow-through. 
10000 rcf, 20 s 

SECTION: RNA purification
25. Add 500 µl of RPE to spin column. Centrifuge at 10000 rcf for 2 minutes and discard flow-through. 
10000 rcf, 2 min 

SECTION: RNA purification
26. Transfer spin columns to fresh collection tubes (without cap)  and centrifuge at max speed (21300 rcf) for 1 min.
21300 rcf, 1 min 

SECTION: RNA purification
27. Transfer spin column to labeled collection tube (with cap). Add 30 µl of RNase-free water directly to the center of the spin column. Centrifuge at 10000 rcf for 1 min. 
10000 rcf, 1 min 

SECTION: RNA purification
28. Pipette the 30 µl flow-through / eluate from each tube back onto the center of its spin column. Centrifuge at 10000 rcf for 1 minute. 10000 rcf, 1 min 

SECTION: RNA purification
29. Discard the spin column. 

Aliquot 2.1 µl of each sample into a PCR tube. Put the rest of the eluted RNA into the -80ºC freezer. 
SECTION: RNA quantification
30. Follow the protocols for the RNA HS Qubit assay. 

Use 1 µl of each sample (and 199 µl of qubit reagent) to quantify. 

Quantities can range from 20 ng/µl to 200 ng/µl depending on the time of year that the samples were collected. 

Record sample concentrations with metadata and extraction #. 
SECTION: RNA Quality Check
31. Follow protocols for the Pico RNA  6000 Bioanalyzer assay 

(reagents are in the "old fridge" and chips are in the drawer in the Apprill Lab (Fye) where the Bioanalyzer lives. 

Dilute all samples to 5 ng / µl before running. 
SECTION: RNA Quality Check
32.