TITLE: RNA Extraction and RIN assessment. 
AUTHORS: Marcos Nascimento

[DESCRIPTION]
Adapted from Mauricio Rodriguez-Lanetty

[GUIDELINES]
Important: 
Work in the fume hood in steps 1-7
Do not worry about RNases in steps 1-5. Your sample is full of them anyway. They are inhibited as long as they are in Trizol. 
From step 6 onwards you should be careful of not contaminating the samples with RNases. Keep cleaning your gloves with RNase Zap (Ambion) through the whole process. The main source of contamination comes from your fingers by accidentally touching the inner part of the tube caps. 
While discarding flow-through in steps 8-12, avoid touching the mouth of the collection tubes with anything!


[STEPS]
SECTION: RNA Extraction
1. Homogenize the starting material using a TissueRuptor and the appropriate volume of Trizol (see table, check Trizol instructions). 

 

SECTION: RNA Extraction
3. Optional: Centrifuge for 10 min at 12,000xg at 4C to eliminate debris and polysaccharides.12000 rcf, 10 min, 4 °C . Collect the supernatant.

SECTION: RNA Extraction
4. Add chloroform to the homogenate (0.2 ml chloroform per ml Trizol used) and shake vigorously for 20 sec, then allow the sample to sit at room temperature for 2-3 min. 3 min 

 


SECTION: RNA Extraction
5. Spin at 10,000g at 4C for 18mins.  10000 rcf, 18 min, 4 °C 

SECTION: RNA Extraction
6. Carefully remove aqueous phase (top) by aspiration (use sterile disposable fine plastic pipette) and transfer to a new sterile RNase-free tube (1.5 ml tube). 
IMPORTANT: Stay away from the aqueous/organic interphase. This is where the DNA and RNases are. It is suggested to sacrifice aqueous material rather than risking taking this precipitate. 
SECTION: RNA Extraction
7. Slowly add an equal volume of 100% RNAse-free EtOH, mixing it as needed. 
SECTION: RNA Extraction
8. Load the sample (up to 700μl) into an RNeasy column (Qiagen kit) seated in a collection tube and spin for 30 sec at 8,000g. Discard flow-through. 
 8000 rcf, 30 s
SECTION: RNA Extraction
10. Add 700µl buffer RW1 onto column and spin 30 sec at 8,000g. Discard flow-through. 8000 rcf, 30 s 

SECTION: RNA Extraction
11. Transfer column into a new 2ml collection tube, add 500µl buffer RPE and spin for 30 sec at 8,000xg. Discard flow-through. Make sure ethanol has been added to the RPE buffer before use. 8000 rcf, 30 s 

SECTION: RNA Extraction
12. Add 500µl buffer RPE and spin 2 min at 8,000xg. Discard flow-through. 8000 rcf, 2 min 

SECTION: RNA Extraction
13. Spin the column for 1 min at 8,000xg to get rid of the remaining buffer in the column. 8000 rcf, 1 min 

SECTION: RNA Extraction
14. Transfer the column to a new 1.5ml collection tube and pipet 30-50µl of RNase-free water directly onto the column membrane. Allow the sample to sit at room temperature for 1-2 min 90 s , and then spin 1 min at 8,000xg to elute RNA. 8000 rcf, 1 min 

SECTION: Analysis on BioAnalyzer
18. Setting up the Chip Priming Station: 
Base plate should be in position C.
Adjust the syringe clip to the top position
SECTION: RNA Extraction
2. Let the homogenate sit at room temperature for 5 min. 5 min 

SECTION: Analysis on BioAnalyzer
19. Clean the Electrodes

SECTION: RNA Extraction
15. Store RNA at -80C if not using it immediately. 
SECTION: Analysis on BioAnalyzer
19.1. Slowly fill one of the wells of the electrode cleaner with 400 μl of fresh RNase-free water. 
SECTION: Analysis on BioAnalyzer
19.2. Open the lid and place the electrode cleaner in the Agilent 2100 Bioanalyzer instrument. Close the lid and leave it closed for 5 minutes. 5 min 

SECTION: Analysis on BioAnalyzer
19.3. Open the lid and remove the electrode cleaner. Wait another 30 seconds to allow the water on the electrodes to evaporate before closing the lid 30 s 

SECTION: Analysis on BioAnalyzer
16. Preparing the Gel. Previously prepared gels can last up to a month in the fridge.
SECTION: RNA Extraction
9. If planning to run the samples on BioAnalyzer right away, move RNApico reagents to room temperature, protecting them from light (~30 mins before use)
SECTION: Analysis on BioAnalyzer
16.1. Place 550 μl of RNA 6000 Pico gel matrix (red ) into the top receptacle of a spin filter.
SECTION: Analysis on BioAnalyzer
16.2. Place the spin filter in a microcentrifuge and spin for 10 minutes at 1500 g 1500 rcf, 10 min 

SECTION: Analysis on BioAnalyzer
16.3. Aliquot 65 μl filtered gel into 0.5 ml RNase-free microcentrifuge tubes that are included in the kit. Store the aliquots at 4 °C and use them within one month of preparation.
SECTION: Analysis on BioAnalyzer
17. Prepare Gel-Dye Mix
SECTION: Analysis on BioAnalyzer
20. Loading the Gel-Dye Mix
SECTION: Analysis on BioAnalyzer
21. Loading the RNA 6000 Pico Conditioning Solution and Marker
SECTION: Analysis on BioAnalyzer
23. Insert chip on BioAnalyzer and select Total RNA Pico as the assay. Select only the wells with Samples to save time.
SECTION: Analysis on BioAnalyzer
24. Remove the chip immediately after the run. Clean electrodes with RNAse-free water.
SECTION: Analysis on BioAnalyzer
17.1. Vortex RNA 6000 Pico dye concentrate (blue ) for 10 seconds and spin down.
SECTION: Analysis on BioAnalyzer
17.2. Add 1 μl of RNA 6000 Pico dye concentrate (blue ) to a 65 μl aliquot of filtered gel (prepared as described in “Preparing the Gel” on Step 16).

SECTION: Analysis on BioAnalyzer
17.3. Cap the tube, vortex thoroughly and visually inspect proper mixing of gel and dye. Store the dye concentrate at 4 °C in the dark again.
SECTION: Analysis on BioAnalyzer
17.4. Spin tube for 10 minutes at room temperature at 13000 g 13000 rcf, 10 min . Use prepared gel-dye mix within one day.

SECTION: Analysis on BioAnalyzer
20.1. Place the chip on the chip priming station. Pipette 9.0 μl of the gel-dye mix at the bottom of the well marked and dispense the gel-dye mix.
 

SECTION: Analysis on BioAnalyzer
20.2. Set the timer to 30 seconds, make sure that the plunger is positioned at 1 ml and then close the chip priming station. The lock of the latch will click when the chip priming station is closed correctly. 

SECTION: Analysis on BioAnalyzer
20.3. Press the plunger of the syringe down until it is held by the clip. Wait for exactly 30 seconds and then release the plunger with the clip release mechanism. 30 s 

SECTION: Analysis on BioAnalyzer
20.4. Visually inspect that the plunger moves back at least to the 0.3 ml mark. Wait for 5 seconds, then slowly pull back the plunger to the 1 ml position.
SECTION: Analysis on BioAnalyzer
20.5. Open the chip priming station and pipette 9.0 μl of the gel-dye mix in each of the wells marked G
 
NOTE: discard the vial with remaining gel-dye mix.
SECTION: Analysis on BioAnalyzer
21.1. Pipette 9 μl of the RNA 6000 Pico conditioning solution (white cap) into the well marked CS. 
 

SECTION: Analysis on BioAnalyzer
21.2. Pipette 5 μL of the RNA 6000 Pico marker (green cap) into the well marked with a ladder symbol and each of the 11 sample wells
 
 

SECTION: Analysis on BioAnalyzer
22. Loading the Diluted Ladder and Samples
SECTION: Analysis on BioAnalyzer
22.1. Before use, thaw ladder aliquots and keep them on ice (avoid extensive warming upon thawing process)
SECTION: Analysis on BioAnalyzer
22.2. Pipette 1 μl of the diluted RNA 6000 Pico ladder into the well marked with the ladder symbol 
 

SECTION: Analysis on BioAnalyzer
22.3. Pipette 1 μl of each sample into each of the 11 sample wells. Add 1 µL of deionized water to each unused sample well. Do not leave any wells empty or the chip will not run properly. 
 

SECTION: Analysis on BioAnalyzer
22.4. Place the chip horizontally in the adapter of the IKA vortex mixer and make sure not to damage the bulge that fixes the chip during vortexing. If there is liquid spill at the top of the chip, carefully remove it with a tissue.
SECTION: Analysis on BioAnalyzer
22.5. Vortex for 60 seconds at 2400 rpm. Make sure that the run is started within 5 minutes.