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

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

[STEPS]
SECTION: Prep
1. Wipe all surfaces inside the RNA hood with:
1. RNAase ZAP (this is a detergent so do not use much as it can leave a soapy residue)
2. RNAse AWAY (this is a weak base, it can be used liberally but does not evaporate quickly)
3. 70% ethanol (etOH), this can be used liberally and evaporates quickly 

use large kimwipes for cleaning surfaces.
SECTION: Prep
6. Set up the tubes you will need for the day by placing them into tube racks in the RNA hood and closing their caps. 
 
Each sample needs: 
    1x bead-bashing tube (from big red qiagen box, in ziplock bag)
    2x RNeasy capped collection tube (from big red qiagen box, in ziplock bag)
    1x RNeasy filter column (from big red qiagen box, pink filter columns in individual blister packs)
    1x Lo-bind 1.5 mL Eppendorf tubes (in ziplock bag inside RNA hood, extras in light-blue Eppendorf box above hood)
SECTION: Cell Lysis
11. Transfer filter tubes for that day's extraction from the –80 freezer into the liquid nitrogen dewar or a cooler with dry ice
(if using dry ice, make sure all samples are in contact with the dry ice)
SECTION: Prep
10. Make fresh 70% ethanol with molecular grade ethanol (in RNA hood) and nuclease-free water (also in RNA hood) in a fresh 15mL falcon tube. (need up to 600 µl per sample)

for 10 samples: 
7000 µl ethanol 
3000 µl water 

Vortex well, then keep in the RNAhood until ready to use later in the protocol

SECTION: Prep
7. Make RLT+BME lysis buffer for the number of samples you have +1 in a fresh 15ml falcon tube: 

Per sample / extra: 
    690 µl of RLT buffer (RLT buffer is in the big red Qiagen box)
    10 µl of BME (BME stays in the chemical hood) 

for 10 samples + 1 extra: 
7590 µl RLT buffer (add this to tube first inside the RNA hood)
110 µl BME (add this to the tube inside the chem hood)

vortex well to mix 

SECTION: Prep
4. Preheat the dry bath thermal block to 65ºC. 
SECTION: Prep
5. Make sure there are enough DNase aliquots for the day's extractions. Move the aliquots you'll need from the freezer to the fridge so they are thawed when you need them. 

!! Each aliquot has 100 µl of DNase. This is exactly enough for 10 samples. !!
SECTION: Cell Lysis
12. Using clean forceps (RNAse-away before first use, etOH between samples) remove filter from cryovial. 

Keeping the filter over a fresh petri-dish half, cut small pieces of filter with clean dissecting scissors (RNase-away before first use, etOH between samples). 

Transfer filter pieces to a bead tube with RLT+BME buffer.

Push the filter pieces down into the beads and buffer so that all filter surfaces remain submerged in the buffer. 

Label the bead tube and put the remaining sample back on dry ice or directly back into the –80 freezer.

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 on one bioanalyzer chip )
SECTION: Cell Lysis
13. Transfer all bead tubes to the thermal block dry bath (use the 2 mL side to make sure tubes are deep enough into the thermal block) and incubate at 65ºC for 10 mins 65 °C 10 min 

SECTION: Cell Lysis
15. Attach the bead tube adapter to the vortex-genie2 (bead tube adapter is in a small navy-blue qiagen box on the top shelf above the bench)

remove the vortex pad by firmly lifting it upward. Line up the notch in the tube adapter with the notch in the vortex connector and press down firmly until you hear/feel a click. 

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

Vortex at the highest speed setting for 5 min 5 min 

SECTION: Cell Lysis
16. Transfer bead tubes to the centrifuge rotor and centrifuge bead tubes for 1 min at 4,000 rcf4000 rcf, 1 min 

SECTION: Cell Lysis
17. Move centrifuge rotor to RNA hood. Using p1000 (BLUE) pipettor set to 1000 µl, remove ALL supernatant from the bead tube and transfer to a matching labeled RNeasy capped collection tube. 

Start by removing liquid above the filter and beads, then "wring-out" the filter 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 some beads and other detritus.  But be careful because too much bead material will get stuck in the pipette tip. 
SECTION: Cell Lysis
18. Centrifuge RNeasy capped collection tubes with supernatant from bead tubes for 1 min at 4,000 rcf 4000 rcf, 1 min 

SECTION: Cell Lysis
19. Move centifuge rotor back into the RNAhood. Transfer 450 µl of supernatant to a clean RNeasy capped collection tube with matching label.  

It is OK to have different volumes from different samples, but it makes it much easier to pick the minimum volume that you are able to recover and transfer the same volume for all the samples. 
SECTION: Cell Lysis
20. Add an equal volume (450 µl) of freshly-prepared 70% EtOH to each sample. Gently and carefully mix by pipette until the density gradient is no longer visible. 

Tranfser 450 of the sample mixed with 70% EtOH to the top of a labeled matching pink spin column. 

(Try to do this with one pipette tip for each sample, add 450 ul of ethanol, mix, transfer 450 ul of sample/ethanol to spin column)
SECTION: RNA purification
21. Transfer spin columns to centrifuge and spin at 10,000 rcf for 20s 10000 rcf, 20 s 

SECTION: RNA purification
22. Discard flowthrough:

Transfer the centrifuge rotor back to RNA hood. Pick up each spin column, separate the collection tube, pour the contents into the waste container, and snap the column back together. Transfer the emptied spin columns to the tube rack. 
SECTION: RNA purification
25. If following along with Qiagen protocol cards, switch over to Part 2 for on-column DNase digestion. 
SECTION: RNA purification
26. Add 350 µl of Qiagen buffer RW1 to the top of each column. Spin at 10,000 rcf for 20 s. 10000 rcf, 20 s 

SECTION: RNA purification
28. Prepare DNase master mix for each sample:

Each sample needs: 
10 µl of DNase stock 
70 µl of RDD Buffer (in blue Qiagen DNase kit box in the door of fridge) 

to make master mix:
Add 700 µl of RDD buffer to 100 µl thawed DNase aliquot. 
Gently pipette up and down to mix. 
Quick spin.
SECTION: RNA purification
29. Add 80 µl of DNase master mix directly to the center of each spin column by pipetting onto the middle of the spin column (not against walls) just above the spin column membrane. !! Do not touch the spin column membrane !!

Incubate for 15 minutes at room temperature. 15 min 

SECTION: RNA purification
30. Add 350 µl of Qiagen buffer RW1 to the top of each spin column. Centrifuge at 10,000 rcf for 20s 10000 rcf, 20 s 

SECTION: RNA purification
32. If following along with Qiagen Protocol cards, go back to part one and restart at step 5. 

Add 500 µl of Qiagen buffer RPE to the top of the spin column. Centrifuge at 100,00 rcf for 20s
10000 rcf, 20 s 

SECTION: RNA purification
34. Add 500 µl of Qiagen buffer RPE to the top of the spin column. Centrifuge at 10000 rcf for 2 minutes. 
10000 rcf, 2 min 

!! Don't forget to change the time on the centrifuge !!
SECTION: RNA purification
36. Centrifuge again at max speed (21,300 rcf) for 1 min.
21300 rcf, 1 min 

!! Don't forget to change the time and speed on the centrifuge !!
SECTION: RNA purification
37. Discard the capless collection tubes and transfer the spin column to the matching labeled 1.5 mL lo-bind eppendorf tube. Add 30 µl of RNase-free water directly to the center of the spin column membrane. Do not touch the filter membrane. 

Centrifuge at 10,000 rcf for 1 min. 
10000 rcf, 1 min 

!! Don't forget to change the speed on the centrifuge !!
SECTION: RNA purification
38. Pipette the 30 µl flow-through / eluate from each tube back onto the center of its spin column filter membrane. Do not touch the filter membrane. 

Centrifuge at 10,000 rcf for 1 minute. 10000 rcf, 1 min 

SECTION: RNA purification
39. Discard the spin column. 

Put RNA samples in the fridge while preparing the Qubit Reagents. 
SECTION: RNA quantification
40. 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 #. 

Enter data into the spreadsheet to determine the dilution factor for the bioanalyzer. 
SECTION: RNA Quality Check
42. Follow protocols for the Pico RNA 6000 Bioanalyzer assay 

Dilute all samples to 5 ng / µl before running. 
SECTION: RNA Quality Check
43. 
 

SECTION: Prep
2. Turn on the UV light (15 minutes, self-timed) in the RNAhood

(don't open the hood during this time, but just in case the UV light will automatically turn off if you do)
SECTION: Prep
3. Wipe surfaces surrounding the RNA hood with:
1. RNAase ZAP (this is a detergent so do not use much as it can leave a soapy residue)
2. RNAse AWAY (this is a weak base, it can be used liberally but does not evaporate quickly)
3. 70% ethanol (etOH), this can be used liberally and evaporates quickly 

Pay attention to surfaces you may rest reagents on, lean against, or touch.
SECTION: Prep
9. add 600 µl of RLT+BME lysis buffer to each bead-bashing tube and close the bead tubes tightly. 

Close the falcon tube containing the remaining RLT+BME tightly. Keep this tube in the RNA hood waste container until all BME garbage is ready to be disposed of.
SECTION: Prep
8. Take out 1 petri dish for every two samples (10 samples = 5 petri dishes). Put petri dishes in the RNAhood. 
SECTION: Cell Lysis
14. During the 10-minute heat incubation, label all other tubes and filter columns to match the sample names on the bead tubes.
SECTION: RNA purification
23. Transfer the remaining 450 µl of sample/ethanol to the top of the matching spin column and centrifuge again at 10,000 rcf for 20 s 10000 rcf, 20 s 

SECTION: RNA purification
27. Discard flow-through by emptying the bottom collection tube into the waste and transferring spin columns to the tube rack.
SECTION: RNA purification
24. Discard flow-through by emptying the bottom collection tube into the waste and then transferring spin columns to the tube rack.
SECTION: RNA purification
31. Discard flow-through by emptying the bottom collection tube into the waste and transferring spin columns to the tube rack.
SECTION: RNA purification
33. Discard flow-through by emptying the bottom collection tube into the waste and transferring spin columns to the tube rack.
SECTION: RNA purification
35. Discard entire collection tube and transfer spin columns to the fresh capless collection tubes (in zip-lock bag in the big red Qiagen box). 
SECTION: Storage + cleanup
41. If samples were successful, aliquot exactly one µl to a PCR tube. Put the PCR tube in a small tube rack box, label it with tape inside the box (the –80 causes the tape glue to fail), and put the box in the –80 freezer. 

Put the remaining sample tubes in the tube box for the project that is already in the –80.  

Empty all tube/tip waste into the garbage and tie up the garbage bag (this is important to contain the BME fumes). 

Wipe down RNA hood and surrounding surfaces with 70% etOH. 

Start the UV light in the hood (double-check that no biological samples or reagents (e.g. enzymes, primers) are in the hood!)

Turn off the centrifuge and heating block. Put away all boxes and reagents.