Oct 31, 2024
Purification of Total RNA from Animal Tissues with QIAcube
This protocol is a draft, published without a DOI.
- Qiagen1
- 1Qiagen
Protocol Citation: Qiagen 2024. Purification of Total RNA from Animal Tissues with QIAcube. protocols.io https://protocols.io/view/purification-of-total-rna-from-animal-tissues-with-dqxd5xi6
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: October 30, 2024
Last Modified: October 31, 2024
Protocol Integer ID: 111301
Abstract
The following protocol describes how to purify total RNA from a maximum of 30 mg fresh or frozen animal tissue as starting material using Qiagen's QIAcube Connect. This protocol requires the RNeasy Mini Kit.
Guidelines
Work RNAse-free and wear gloves.
Materials
Materials
- maximum amount of 30 mg fresh or frozen tissue or 15–20 mg RNA protect stabilized tissue
Consumables
- β-mercaptoethanol (BME)
- 100% ethanol
- HyPure or Nucleus Free Water (NFW)
- RNAse-Free DNase Set
- RNase Free Water
- Qubit Fluorometer
- Qubit Assay Tubes
- 1.5 mL Tubes
- 2 mL Tubes
- RNeasy Plus Mini Kit
- 7mm Steel Beads
- 15 mL Conical Tube
- 1 mL Plastic Syringe
- 25G x 1 1/2 Sterile Needle
- Qubit RNA HS Assay Kit
Equipment
- Ice bucket with ice
- Dry ice
- Timer
- Microfuge
- Vortex mixer
- Qubit fluorometer (or equivalent for QC check)
- QIAcube Connect
- Qiagen TissueLyse II
- Pipettes and pipette tips P2, P10, P20, P100, P200, P1000
Making lysis buffer master mix
Making lysis buffer master mix
Make a master mix consists of RLT and BME. The volumes of each reagent depends on how many samples are being processed at once.
Note
When make master mixes, add 0.5 to your sample number so that you have a bit of extra reagent in case of mistakes.
Example: if you have 6 samples, do your calculations with 6.5
Combine the following two reagents in a 5mL or 15mL conical tube. Make sure to dispense in a fume hood and wear appropriate protective clothing.
General volumes based on tissue type:
- Hippocampus tissue: 400uL of RLT buffer + 0.4uL β-mercaptoethanol per sample
- Cortex tissue: 1 mL of RLT buffer + 10uL β-mercaptoethanol per sample
Safety information
This master mix can be stored/used for up to a month after mixing. Because of this, make sure that you clearly label the name of the reagent and the date made on the side of the tube.
Making fresh 70% ethanol
Making fresh 70% ethanol
Make equal amounts of fresh 70% EtOH for homogenate. Depending on the weight of the sample, the total volume will change. Consider making 3 mL to 5 mL.
Dissolving DNase I and Making DNase I + RDD mixture
Dissolving DNase I and Making DNase I + RDD mixture
Using a P1000 pipette, transfer 550uL of RNase free water to a clean 1.5mL tube and set aside for the next step.
Take the DNase I vial and gently tap the base on the bench a few times to collect the powder to the bottom of the vial.
Carefully, lift the metallic flap off the top of the DNase I vial, exposing the gray rubber stopper.
Assemble the needle and syringe and use it to collect all of the RNase free water. Carefully, insert the needle about halfway into the stopper and add the 550uL water to the powder.
Close the metallic flap then swirl around until powder is dissolved and invert a few times. Carefully, remove the metal stopper. Remove the rubber stopper and aliquot solution into clean 2mL tubes.
Use the table below to make master mixes. Note that you can only thaw the DNase/RDD mixture once, so account for how many working tubes you will be processing at once.
| Number of Samples | uL of DNase I | |
| 2 | 27uL | |
| 3 | 37uL | |
| 4 | 48uL | |
| 5 | 59uL | |
| 6 | 70uL | |
| 7 | 81uL | |
| 8 | 91uL | |
| 9 | 102uL | |
| 10 | 113uL | |
| 12 | 135uL |
Safety information
These aliquots can only be stored for up to 9 months in -20°C or 6 weeks in 2–8°C
Label the tubes properly. (i.e. reagent name, date made, for how many samples)
Store the extra aliquots in -20ºC. Keep your working aliquot on ice.
Based on the number of samples you are processing, follow the table below to find the volume of RDD buffer that you should add to your working aliquot of DNase I.
| Number of Samples | uL of RDD | |
| 2 | 186uL | |
| 3 | 263uL | |
| 4 | 338uL | |
| 5 | 413uL | |
| 6 | 489uL | |
| 7 | 564uL | |
| 8 | 640uL | |
| 9 | 716uL | |
| 10 | 791uL | |
| 12 | 942uL |
Mix by pipetting and keep on ice. Do NOT vortex
Preparing samples
Preparing samples
Place bag of 7mm steel beads on dry ice. Leave it there for at least 15 minutes.
Note
Make sure that your tissue samples are in 2mL tubes and on dry ice.
Only start once steel beads have chilled for full time.
At the bench, use tweezers to add one cold 7mm steel bead from the bag to each tube. Keep sample tubes on dry ice.
Take the dry ice box (with samples), and a tube rack to the chemical hood.
Under the chemical hood add 400uL of previously made lysis buffer to each tube and place on rack. At this point, it is okay if the samples are in room temperature but you want to move onto the next step as fast as possible.
Immediately transfer the samples to Qiagen's TissueLyser holder. Make sure that you split the samples so the holder is balanced.
Place the holder in the TissueLyser II machine and set to 30Hz for 2 minutes. Run machine.
While tubes are still in the TissueLyser holder, take the samples to the cold room and use the centrifuge in there to spin down the homogenate (tissue+lysis mixture) for 3 minutes at full speed.
- During this time, you can move onto the "Preparing the QIAcubes" section. If you are unable to finish it before these 3 minutes are done, do so after your samples are ready.
With a P1000 pipette, transfer 350uL of the homogenate to a new, labeled 2mL tube and proceed to the next part. Make sure your samples are on ice.
Discard empty 2mL tube and steel bead in waste basket. Waste should be dumped in special container outside of chemical hood.
Preparing the QIAcubes
Preparing the QIAcubes
Based on your sample ID's, label the rotor adaptors, 1.5mL collection tubes and the lids of the pink RNase mini spin column.
Check the reagent bottles in the QIAcube. If any of the bottles are less than a third full, you need to top off with the appropriate buffer.
Dump any remaining 70% ethanol in bottles and replace with freshly made 70% ethanol.
Loading the QIAcubes
Loading the QIAcubes
Turn on the machine and login if necessary. Assign a QIAcube position with each of your samples.
Load the labeled 2mL tube containing 350uL of the sample homogenate into the QIAcube shaker plate and its corresponding rotor, spin column and collection tube in the rotator. Make sure the position numbers match with the assigned sample.
Setup the machine with the right protocol: "RNA RNeasy mini > Animal tissue & cell - DNase digest"
When prompted on the screen, set up the reagents bottles so that they match what is shown on the screen.
Place the 1000uL tips in the correct holder spot in the machine.
Place the tube of DNase I + RDD mixture into the correct holder spot in the machine.
Do a once over. Make sure that everything is in the correct spot and that all tubes are firmly in place. When ready, press "start".
After the QIAcube
After the QIAcube
When the QIAcube is done, take out the rotor adapters. Check to make sure each collection tube has some elution.
- If there is no elution, save the spin column so you can try and get the sample out manually.
Place collection tubes + sample on ice and take back to the bench.
Dump the liquid in the rotor adaptors into the liquid chemical waste container in the chemical hood and discard all rotor adaptors and spin columns in the hazardous solid waste container next to the chemical hood.
Check RNA concentration using Qubit or an equivalent.
Qubit protocol
Qubit protocol
Make a Master Mix:
Based on the number of samples, mix together the proper volume of buffer and dye.
Note
Values can be calculated or found on Qubit chart at the bench
RNA HS Buffer = 198 uL x (# sample + 0.5)
RNA HS Dye = 1 uL x (# sample + 0.5)
Mix by vortexing. Place in drawer or cover to avoid light exposure until use.
Dilute RNA if necessary (1:10 dilution).
Using a P200, transfer 199 uL of the master mix into each labeled qubit tube.
Mix (diluted) RNA samples with a P20.
Using a P2.5, transfer 1 uL of the diluted RNA to the corresponding qubit tube.
Vortex for 2-3 seconds. Incubate for 2 minutes.
Measure the concentration using the Qubit.
Protocol references
Acknowledgements
The protocol reproduced here is originally provided by Qiagen along with the RNeasy Mini Kit. Original protocol can be found in the RNeasy Mini Handbook linked here: https://www.qiagen.com/us/resources/resourcedetail?id=f646813a-efbb-4672-9ae3-e665b3045b2b&lang=en