Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps

Kit Contents

Dynabeads® Oligo (dT)₂₅ *   1 × 2 ml
Binding Buffer            4 ml
Washing Buffer B       4ml
Elution Buffer             4 ml

*Approximately 5 mg/ml, supplied in PBS pH 7.4, containing 0.02% NaN₃ as a preservative

Product Description

This product has been designed for rapid isolation of highly purified and intact mRNA from total RNA.

Principle of Isolation

The isolation protocol relies on base pairing between the poly A residues at the 3’ end of most mRNA and the oligo (dT)₂₅  residues covalently coupled to the surface of the Dynabeads® . Other RNA species lacking a poly A tail will not hybridize to the beads and are readily washed away. 1 mg of beads (200 µl) will isolate up to µg of mRNA, depending on the sample.  A typical mammalian cell contains about 10-30 pg of total RNA, from which 1-5% is mRNA.

Physical Characteristics of Dynabeads® Oligo (dT)₂₅

Diameter:    2.8 µm ± 0.2 µm
                     (C.V. max 5%)
Surface area:   3–7 m² /g
Density:  approx. 1.6 g/cm³
Magnetic mass susceptibility:   20 ± 25 × 10–6 m³/kg

Buffers

Binding Buffer            4 ml

20 mM Tris-HCl (pH 7.5)
1.0 M  LiCl
2 mM  EDTA

Washing Buffer B      4 ml

10 mM Tris-HCl (pH 7.5)
0.15 M  LiCl
1 mM  EDTA
10 mM Tris-HCl (pH 7.5)  4 ml

Additional Material Required

• Magnet: See magnets-selection for magnet recommendations.
• Mixer allowing both tilting and rotation
• Sterile, RNase free microcentrifuge tubes
• Sterile, RNase free pipette tips

Technical Advice

• Work RNase free and wear gloves.


• Keep Dynabeads® Oligo (dT)₂₅ in liquid suspension during storage and all handling steps. Resuspend well before use.


• All common buffers for mRNA purification and isolation can be used with Dynabeads® Oligo (dT)₂₅.

Purification of mRNA from total RNA

The protocol below describes mRNA isolation from 75 μg of total RNA as starting material.

Preparation of RNA

1. Adjust the volume of your 75 μg total RNA 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 also step 3 under ‘Preparation of Dynabeads® ’).


2. Heat to 65°C for 2 minutes to disrupt secondary structures. Place on ice.

Preparation of Dynabeads®

1. Transfer 200 μl (1 mg) of well resuspended Dynabeads® to a microcentrifuge tube. Place the tube on the magnet for 30 seconds, or until all Dynabeads® have migrated to the tube wall.


2. Pipette off the supernatant, remove the tube from the magnet and add 100 μl Binding Buffer to calibrate the beads. Put the tube back on the magnet and remove the supernatant. Remove the tube from the magnet.


3. Add 100 μl Binding Buffer to the Dynabeads® . Optimal hybridization conditions are obtained in Binding Buffer added in a 1:1 ratio relative to sample volume. If your total RNA is more dilute than 75 μg/100 μl, then simply add an equal volume ofBinding Buffer to the Dynabeads® .

Isolation of mRNA

1. 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 mRNA to anneal to the oligo (dT)₂₅ on the beads.


2. Place the tube on the magnet until solution is clear. Remove the supernatant.


3. Remove the tube from the magnet and wash the mRNA-bead complex twice with 200 μl Washing Buffer B. Remove all the supernatant between each washing step with the help of the magnet – this is important when working with small volumes.


4. If elution is required, add desired amount (10–20 μl, or down to 5 μl) of 10 mM Tris-HCl, pH 7.5. Heat to 65–80°C for 2 minutes and place the tube immediately on the magnet. Transfer the eluted mRNA to a new RNase-free tube. Note:  This protocol can be scaled up or down to adjust mRNA yield. Increase or decrease the quantities of the kit reagents proportionally with any changes in total RNA starting sample. Optimization may be needed.

Regeneration and Reuse of Dynabeads® Oligo (dT)₂₅

The oligo (dT)₂₅ sequences are covalently attached to the bead surface. Whilst this enables a single step hybridization, the covalent nature of the binding also allows regeneration of the Dynabeads® Oligo (dT)₂₅. The beads may be reused a total of four times without loss of yield. The reusable properties of these Dynabeads® provides a cost effective choice for the user. When mRNA is to be isolated from the same crude extract, the Dynabeads® Oligo (dT)₂₅ can be reused without regeneration. By reusing the Dynabeads® on the same sample (without regeneration), large amounts of mRNA can be isolated. Simply follow the mRNA isolation procedure. After elution of the mRNA, wash the Dynabeads® once in Binding Buffer before adding the sample and a new capture of mRNA is performed. This can be repeated several times until no mRNA is left in the sample.


Note:  The buffers in this kit is not sufficient for re-use of the Dynabeads® , and they will have to be made. The Binding Buffer in this kit is supplied as a 2 × concentrate. This is to obtain optimal hybridization condition when mixing 1:1 with the sample. When used to wash the Dynabeads® for reuse, adjust the Binding Buffer to 1 × concentration by adding an equal volume of DEPC-treated water.

Note:   Buffers such as Tris cannot be DEPC treated as Tris inactivates DEPC. Solutions should be DEPC treated and autoclaved before adding Tris. Subsequently after adding Tris, autoclave the solution again. DEPC is a suspected carcinogen – handle with care. Wear gloves.

Regeneration Protocol

Use this protocol if you need to avoid cross contamination of mRNA between samples.

1. Resuspend used Dynabeads® (original volume 200 μl) in 200 μl Reconditioning Solution and transfer suspension to a new RNase-free tube.


2. Incubate at 65°C for 2 minutes.


3. Place tube in magnetic field for at least 30 seconds and remove supernatant.


4. Wash twice in Reconditioning Solution, by repeating steps 1 and 3 twice.


5. Resuspend Dynabeads® in 200 μl Storage Buffer Oligo (dT)₂₅.


6. Place tube on the magnet for at least 30 seconds and remove supernatant.


7. Repeat steps 5 and 6 three times, or until pH of supernatant is below 8.0.


8. Resuspend Dynabeads®   in desired volume of Storage Buffer Oligo (dT)₂₅. The Dynabeads® are now reconditioned and ready for another mRNA isolation. Store Dynabeads® in Storage buffer Oligo (dT)₂₅ at 2–8°C.

Buffers for regeneration(not supplied):

Reconditioning Solution:

0.1 M NaOH

Storage Buffer Oligo (dT)₂₅:  

250 mM Tris-HCl (pH 7.5)
20 mM EDTA
0.1% Tween20
0.02% Sodium azide (NaN₃)

Note:   Do not mix regenerated Dynabeads® Oligo (dT)₂₅ with original stock suspension.

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

This product is stable until the expiry date stated on the label, when stored unopened at 2–8°C. Store opened vials at 2–8°C and avoid bacterial contamination. Do not store the Dynabeads® Oligo (dT)₂₅ in distilled water.

Technical Support

Certificate of Analysis/Compliance is available upon request. The latest revision of the package insert/instruction for use is available on www.lifetechnologies.com.

Warnings and Limitations

The Dynabeads® mRNA Purification Kit is for research use only. The product is not for use in human diagnostic or therapeutic procedures. The Dynabeads® Oligo (dT)₂₅ contains 0.02% sodium azide (NaN₃) as a preservative. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build-up. Preservatives such as sodium azide are toxic if ingested. Avoid pipetting by mouth. Standard methods for preventing contamination by RNases during the preparation of mRNA must be taken. Take precautions to prevent RNase contamination of opened vials. Material Safety Data Sheet is available from www.lifetechnologies.com.