Mar 12, 2024
RNA Extraction and RIN assessment.
This protocol is a draft, published without a DOI.
- 1UCSF
Protocol Citation: Marcos Nascimento 2024. RNA Extraction and RIN assessment. . protocols.io https://protocols.io/view/rna-extraction-and-rin-assessment-bvbin2ke
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: May 26, 2021
Last Modified: March 12, 2024
Protocol Integer ID: 50250
Disclaimer
Protocol Adapted from Mauricio Rodriguez-Lanetty and Qiagen's RNA Pico 6000 user manual
Abstract
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!
Materials
RNeasy Mini KitQiagenCatalog #74104
ChloroformFisher ScientificCatalog #BP1145-1
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Equipment
new equipment
NAME
Bioanalyzer 2100 instrument
BRAND
G2939BA
SKU
with RNA 6,000 Pico LabChip kit
SPECIFICATIONS
Equipment
TissueRuptor II
NAME
Qiagen
BRAND
9002755
SKU
RNA Extraction
RNA Extraction
36m
Homogenize the starting material using a TissueRuptor and the appropriate volume of Trizol (see table, check Trizol instructions).
| A | B | |
| Amount of Tissue | Trizol Volume | |
| 100mg | 1ml | |
| 50mg | 0.5ml |
Let the homogenate sit at room temperature for 5 min. 00:05:00
5m
Optional: Centrifuge for 10 min at 12,000xg at 4C to eliminate debris and polysaccharides.12000 rcf, 4°C, 00:10:00 . Collect the supernatant.
10m
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. 00:03:00
| A | B | |
| Trizol used in step 1 | Chloroform | |
| 1ml | 200ul | |
| 0.5ml | 100ul |
3m
Spin at 10,000g at 4C for 18mins. 10000 rcf, 4°C, 00:18:00
18m
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.
1m
Slowly add an equal volume of 100% RNAse-free EtOH, mixing it as needed.
5m
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, Room temperature, 00:00:30
30s
If planning to run the samples on BioAnalyzer right away, move RNApico reagents to room temperature, protecting them from light (~30 mins before use)
Add 700µl buffer RW1 onto column and spin 30 sec at 8,000g. Discard flow-through. 8000 rcf, Room temperature, 00:00:30
30s
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, Room temperature, 00:00:30
30s
Add 500µl buffer RPE and spin 2 min at 8,000xg. Discard flow-through. 8000 rcf, Room temperature, 00:02:00
2m
Spin the column for 1 min at 8,000xg to get rid of the remaining buffer in the column. 8000 rcf, Room temperature, 00:01:00
1m
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 00:01:30 , and then spin 1 min at 8,000xg to elute RNA. 8000 rcf, Room temperature, 00:01:00
2m 30s
Store RNA at -80C if not using it immediately.
Analysis on BioAnalyzer
Analysis on BioAnalyzer
27m
Preparing the Gel. Previously prepared gels can last up to a month in the fridge.
Place 550 μl of RNA 6000 Pico gel matrix (red ) into the top receptacle of a spin filter.
Place the spin filter in a microcentrifuge and spin for 10 minutes at 1500 g 1500 rcf, 00:10:00
10m
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.
5m
Prepare Gel-Dye Mix
Vortex RNA 6000 Pico dye concentrate (blue ) for 10 seconds and spin down.
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).
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.
Spin tube for 10 minutes at room temperature at 13000 g 13000 rcf, Room temperature, 00:10:00 . Use prepared gel-dye mix within one day.
10m
Setting up the Chip Priming Station:
- Base plate should be in position C.
- Adjust the syringe clip to the top position
1m
Clean the Electrodes
Slowly fill one of the wells of the electrode cleaner with 400 μl of fresh RNase-free water.
Open the lid and place the electrode cleaner in the Agilent 2100 Bioanalyzer instrument. Close the lid and leave it closed for 5 minutes. 00:05:00
5m
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 00:00:30
30s
Loading the Gel-Dye Mix
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.
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.
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. 00:00:30
30s
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.
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.
Loading the RNA 6000 Pico Conditioning Solution and Marker
Pipette 9 μl of the RNA 6000 Pico conditioning solution (white cap) into the well marked CS.
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
Loading the Diluted Ladder and Samples
Before use, thaw ladder aliquots and keep them on ice (avoid extensive warming upon thawing process)
Pipette 1 μl of the diluted RNA 6000 Pico ladder into the well marked with the ladder symbol
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.
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.
Vortex for 60 seconds at 2400 rpm. Make sure that the run is started within 5 minutes.
Insert chip on BioAnalyzer and select Total RNA Pico as the assay. Select only the wells with Samples to save time.
Remove the chip immediately after the run. Clean electrodes with RNAse-free water.