Oct 30, 2024
Dynabeads mRNA Purification
This protocol is a draft, published without a DOI.
- Invitrogen1
- 1Invitrogen
External link: https://www.thermofisher.com/order/catalog/product/61006
Protocol Citation: Invitrogen 2024. Dynabeads mRNA Purification. Protocol exchange https://protocols.io/view/dynabeads-mrna-purification-dqww5xfe
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 30, 2024
Protocol Integer ID: 111286
Keywords: polyA selection, mRNA purification, oligodT selection, dynabeads
Abstract
The following protocol describes mRNA isolation using 75 μg of total RNA as starting material. This protocol can be scaled up or down by increasing or decreasing reagent volumes proportionally with any changes in the amount of the total RNA starting material. Optimization may be needed.
Attachments
Guidelines
- Work RNAse-free and wear gloves.
- Keep the Dynabeads‱ Oligo (dT)25 in liquid suspension during storage and handling steps. Resuspend well before use.
- All common buffers for mRNA purification and isolation can be used with Dynabeads‱ Oligo (dT)25
Materials
Kit contents:
- Dynabeads‱ Oligo (dT)25
- Binding Buffer
- Washing Buffer B
- 10 mM Tris-HCl
User provided materials:
• DynaMag‱ Magnet (See thermofisher.com/magnets for recommendations)
• Sample mixer allowing tilting and rotation of tubes (e.g. HulaMixer‱ Sample Mixer)
• Sterile RNase-free microcentrifuge tubes
• Sterile RNase-free pipette tips
Safety warnings
Dynabeads‱ Oligo (dT)25 contains 5 mg/mL of beads in phosphate buffered saline (PBS), pH 7.4, with 0.05% Tween‱ and 0.02% sodium azide as a preservative. Caution: Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. See "Description of materials" for Buffer content.
Prepare RNA
Prepare RNA
Adjust the volume of the total RNA sample (75 μg) to 100 μL with distilled DEPC-treated water, or with 10 mM Tris-HCl, pH 7.5. Omit this step if only a small adjustment is needed (see step 6 of “Prepare Dynabeads‱ magnetic beads”).
Heat the sample to 65°C for 2 minutes to disrupt secondary structures.
Place sample on ice.
Prepare Dynabeads™ magnetic beads
Prepare Dynabeads™ magnetic beads
Transfer 200 μL (1 mg) of resuspended Dynabeads‱ magnetic beads to a microcentrifuge tube. Place the tube on the magnet for 30 seconds, or until all the Dynabeads‱ magnetic beads have adhered to the tube wall.
Discard the supernatant, remove the tube from the magnet, and add 100 μL of Binding Buffer to equilibriate the beads. Place the tube on the magnet and remove the supernatant. Remove the tube from the magnet.
Add 100 μL Binding Buffer to the Dynabeads‱ magnetic beads. Optimal hybridization conditions require a 1:1 ratio of Binding Buffer to sample volume. If the total RNA is more dilute than 75 μg/100 μL, then add a volume of Binding Buffer equal to the sample volume to the Dynabeads‱ magnetic beads.
Isolate mRNA
Isolate mRNA
Add the total RNA to the Dynabeads‱/Binding Buffer suspension. Mix thoroughly, and rotate on a roller or mixer for 3–5 minutes at room temperature to allow the mRNA to anneal to the oligo (dT)25 on the beads.
Place the tube on the magnet until the solution is clear. Remove the supernatant.
Remove the tube from the magnet and wash the mRNA-bead complex twice with 200 μL Washing Buffer B. Use the magnet to remove all traces of supernatant between each washing step (this is important when working with small volumes).
(Optional) Add 10–20 μL (or down to 5 μL) of 10 mM Tris-HCl, pH 7.5 to elute the mRNA.
Heat the sample at 65°C to 80°C for 2 minutes and place the tube immediately on the magnet.
Transfer the eluted mRNA to a new RNase-free tube.
Acknowledgements
The protocol reproduced here is originally provided by Invitrogen (Thermo Fisher) along with the Dynabeads‱ mRNA Purification Kit (cat. #61006). Original protocol is attached as a pdf.