Introduction

Intended Use

Dynabeads® Oligo (dT)25 are designed for the rapid isolation of highly purified, intact mRNA from eukaryotic total RNA or directly from crude extracts of cells, animal and plant tissues. The isolated mRNA can be used directly in most downstream applications in molecular biology: RTPCR, solid-phase cDNA library construction, S1 nuclease analysis, ribonuclease protection assay, primer extension, dot and slot hybridization, in vitro translation experiments, RACE, subtractive hybridization, northern analysis, gene cloning and gene expression analysis.

Principle

The use of Dynabeads® Oligo (dT)25 relies on base pairing between the poly A tail of messenger RNA and the oligo dT sequences bound to the surface of the beads. After annealing, the vial is placed on a magnet (Dynal® MPC™) to concentrate the beads with their bound mRNA at the side of the tube. The supernatant containing unwanted contaminants is discarded. The protocol can be performed in 15 minutes, without the need to prepare total RNA or perform any other purification steps. The oligo dT bound to the bead surface can be used to both capture the mRNA and act as a primer for reverse transcriptase during first strand cDNA synthesis. As the oligo dT  is covalently bound to the Dynabeads® surface it is possible to regenerate the Dynabeads® Oligo (dT)25 for reuse.

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Protocols

Additional protocols to those presented here, as well as technical information and literature are available upon request.

Dynabeads® Washing Procedure

This procedure takes approximately 10 minutes and can be carried out at a convenient interval during the mRNA purification.

  1. Resuspend the Dynabeads® Oligo (dT)25 thoroughly in the vial to obtain a uniform brown suspension and transfer 200 μl (1 mg) of beads to a tube.

  2. Place the tube on a magnet (Dynal® MPC™) for 1-2 min. The Dynabeads® Oligo (dT)25 will migrate to the side of the tube nearest the magnet.

  3. Remove the supernatant with a pipette while the tube remains on the magnet.

  4. Remove the tube from the magnet and add 100 μl Binding Buffer to resuspend the beads. Again place the tube on the magnet for 1-2 min.

  5. Remove the supernatant while the tube remains on the magnet.

  6. Resuspend the beads in 100 ul Binding Buffer.


Preparation of lysate from animal tissues, plants and cells

The protocol is recommended for a sample size of 20-50 mg animal tissue, 100 mg plant tissue or 1-4 x 106 cells, but can be scaled up or down to suit specific sample size requirements.

Preparation of lysate from solid animal and plant tissues

  1. Aliquot the amount of animal or plant tissue while it is frozen. Use the specified amount of tissue, as an excess of tissue will reduce the mRNA yield and purity.

  2. Grind frozen tissue in liquid nitrogen. Ensure tissue remains frozen at all times to avoid RNA degradation.

  3. Transfer the frozen powder to a homogenizer containing 1 ml Lysis/Binding Buffer and homogenize for 1-2 min until the tissue has completely lysed. A rapid lysis in Lysis/Binding Buffer will prevent degradation of mRNA. If the raw extract is noticeably viscous a shear step might be helpful.

  4. Spin the lysate for 30-60 seconds in a microcentrifuge to remove debris. The lysate is now ready for mRNA isolation, or can be frozen and stored at -80°C for later use.


Preparation of lysate from cultured cells and cell suspensions

  1. Wash the cell suspension in phosphate-buffered saline (PBS) and centrifuge to obtain a cell pellet. The cell pellet can be used immediately, or frozen in liquid nitrogen and stored at -80°C for later use. A stored cell pellet should be used directly from frozen.

  2. Add 1.0 ml Lysis/Binding Buffer to the cell pellet (1-4 x 106 cells). Pipette up and down a couple of times to ensure complete lysis. The release of DNA during lysis results in a viscous solution which confirms complete lysis.

  3. Reduce the viscosity by a DNA-shear step. The lysate is passed three times through a 21 gauge needle using   1-2 ml syringe. Repeated shearing may cause the lysate to foam. Foaming should however not affect the mRNA yield. The foam can be reduced by a 30 second centrifugation.

  4. The lysate is now ready for mRNA isolation or can be frozen and stored at -80°C for later use.


Isolation of mRNA from crude lysate

  1. Remove the solution from the washed Dynabeads® Oligo (dT)25 and add the lysate

  2. Mix beads and lysate. Anneal by rotating on a mixer for 3-5 min at room temperature, increase the annealing time if the solution is viscous. During this step the mRNA anneals to the oligo dT sequence.

  3. Place the vial on the magnet for 2 min and remove the supernatant.

  4. Wash the beads twice using the magnet to separate the beads from the supernatant, first with Washing Buffer A (0.5 - 1 ml) and then with Washing Buffer B (0.5 - 1 ml). Washing is performed at room temperature. The Dynabeads® Oligo (dT)25 must be mixed thoroughly in the Washing Buffer to remove contaminants which may have bound non-specifically. Make sure to remove the supernatant completely between washing steps.

  5. If the isolated mRNA is to be used in enzymatic downstream applications whilst still bound to the beads (e.g. solid-phase cDNA synthesis), wash one extra time with Washing Buffer B (500 μl) followed by one wash with the enzymatic buffer to be used in the downstream application.

  6. If the mRNA is to be eluted from the beads, first remove the final Washing Buffer B and add 10 - 20 μl 10 mM Tris-HCl. Incubate at 75 - 80°C for 2 min, then place the tube on the magnet and quickly transfer the supernatant containing the mRNA to a new RNase-free tube. The final yield may vary somewhat between tissues/cells depending on mRNA abundance.


Purification of mRNA from total RNA

In the below example, mRNA is purified from 75 μg of total RNA starting material.

  1. Adjust the volume of the 75 μg total RNA sample to 100 μl with distilled DEPC-treated water or with 10 mM Tris-HCl pH 7.5.

  2. Add 100μl of Binding Buffer. If total RNA is more dilute than 75 μg/100 μl, then simply add an equal volume of Binding Buffer to the beads.

  3. Heat to 65°C for 2 min to disrupt secondary structures. Immediately place on ice.

  4. Add the 200 μl of total RNA to the 100 μl washed beads. For every 75 μg total RNA use 1 mg beads which have been washed and resuspended in 100 μl of Binding Buffer.

  5. Mix thoroughly and anneal by rotating continuously on a mixer for 5 min at room temperature.

  6. Place the tube on the magnet for 1-2 min and carefully remove all the supernatant.

  7. Remove the tube from the magnet and add 200 μl Washing Buffer B. Mix by pipetting carefully a couple of times. Again use the magnet to pull the beads to the side of the tube. Carefully remove all the supernatant.

  8. Repeat the washing step as described in step 7.

  9. If the mRNA does not need to be eluted off the beads, wash one more time using the same buffer that will be used in the downstream application, e.g. reverse transcription first strand synthesis buffer (without the enzyme). Then resuspend the beads in an appropriate volume of the downstream buffer.

  10. If mRNA elution is required, add the desired amount (10 - 20 μl) of cold 10 mM Tris-HCl. Heat to 75-80°C for 2 min and place the tube immediately on the magnet. Quickly transfer the eluted mRNA to a new RNase-free tube.


Regeneration and reuse of Dynabeads® Oligo (dT)25

The oligo (dT) sequences are covalently attached to the surface of the Dynabeads® . This enables singlestep hybridization, and also allows regeneration of the beads for multiple use. The beads may be reused a total of four times without loss of yield. When mRNA is isolated from the same sample, the beads can be reused without regeneration.

Regeneration of Dynabeads
® Oligo (dT)25

To avoid carry-over of mRNA between different samples the beads should be washed three times in 200 μl Reconditioning Solution by standard magnetic separation. Incubate at 65°C for 2 minutes at the first wash. Then wash using 200 μl Storage Buffer Oligo (dT)25 and continue carrying out washes until the pH is below 8.0. Resuspend the beads in the desired volume of Storage Buffer Oligo (dT)25. The beads are now regenerated and ready for mRNA isolation. Store the beads at 2-8°C. Do not mix regenerated beads with the original stock suspension

Reuse on the same sample

By reusing the Dynabeads® Oligo (dT)25 on the same sample (without regenerating the beads) larger amounts of mRNA can be isolated. After elution of mRNA, wash the beads (original volume 200 μl) once in Lysis/Binding Buffer (300 μl). Then add the beads back to your sample for further mRNA isolation. Isolation can be repeated several times until all the mRNA is captured from the sample.

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Technical Advice

Preparation of mRNA for downstream applications

For northern analysis, the mRNA can be eluted directly into a loading buffer containing formamide and loaded directly onto the gel. if the mRNA is to be used in downstream enzymatic applications (cDNA synthesis, in vitro translations experiments, RT-PCR etc.), detergents should be omitted in the final washing steps and the elution step. Enzymatic downstream applications are not inhibited by the presence of the beads. It is possible to construct solid-phase cDNA libraries specific for a particular cell type or tissue directly on the bead-surface. The covalently linked oligo dT sequence is used both to capture the mRNA and as a primer for the reverse transcriptase to synthesize the first strand cDNA. This results in a covalently linked first-strand cDNA library.

General recommendations

  • Before use, resuspend the Dynabeads® Oligo (dT)25 well to obtain a homogeneous dispersion of beads in solution.
  • Keep the beads in liquid suspension during storage and all handling steps. Beads that are left unsuspended for a long period will dry out, leading to a possible reduction in isolation efficiency.
  • The complete removal of all the buffer during washing is extremely important when working with small volumes.
  • When working with cells isolated by immunomagnetic separation (IMS) using cell-specific Dynabeads® , ensure that all IMS-Dynabeads® are removed from the lysate before adding Dynabeads® Oligo (dT)25.
  • We recommend the Dynabeads® -mRNA complex to be used immediately for RT-PCR. If storage is necessary, elute the mRNA from the beads and freeze.
  • RNases are very stable, active enzymes and generally require no cofactors to function. RNase inhibitors may be added to the protocol at any step, although this is normally not necessary. If storage of the eluted mRNA is required, addition of an RNase inhibitor at the elution step is recommended.


Please contact Invitrogen Dynal® for further technical information

Avoiding contamination

To obtain good preparations of eukaryotic mRNA, it is necessary to minimize the activity of RNases by creating a ribonuclease-free environment. The following precautions should be taken to avoid contamination: Contamination by personnel Your hands are a major source of contaminating RNases. Disposable gloves should be worn at all times during the procedure. Gloves remain RNase free only if they do not come into contact with "dirty" glassware and surfaces. Change gloves frequently when working with RNA.

Solutions

Any water and salt solutions used in RNA preparation should be RNase free, i.e. by treatment with diethylpyrocarbonate (DEPC). Wherever possible, the solutions should be treated with 0.1% DEPC for at least 1 hour at 37°C and then heated to 100°C for 15 minutes or autoclaved for 15 minutes to remove any traces of DEPC. Tris Buffers cannot be DEPC-treated, as Tris inactivates DEPC. Solutions should be DEPC-treated and
autoclaved before adding Tris. After addition of Tris, the solution should be autoclaved again. DEPC is a suspected carcinogen and should be handled with great care.

Plasticware

Sterile, disposable plasticware is essentially free of RNases and can be used for the preparation and storage of RNA without pre-treatment. General laboratory plasticware should be rinsed with chloroform.

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General Information

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO n13485:2003.

Description of Material

Dynabeads® Oligo (dT)25 are uniform superparamagnetic, monodisperse polymer particles with oligo dT sequences covalently coupled to the bead surface. The beads are supplied as a suspension of approximately 5 mg/ml in phosphate-buffered saline (PBS) pH 7.4 containing 0.02% NaN3 as a preservative. The product is available in three formats: 2 x 1 ml (Cat. no. 610.02), 5 x 1 ml (Cat no. 610.05) and 5 x 10 ml (Cat no. 610.50)

Product Characteristics

Typical bead characteristics for any given lot of this product:
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: 120 ± 25 x 10-6 m3/kg
Certificate of Analysis (CoA) is available upon request.

Binding Capacity

Up to 2μg poly A+ RNA can be isolated per 200 μl (1 mg) of beads, depending on the tissue or cell type and the expression level of the mRNA. A typical mammalian cell contains about 10-30 pg of RNA of which 1 - 5% is mRNA  The total capacity per ml of beads is approx. 10 μg mRNA. If the same beads are reused for a total of 5 mRNA isolations (four regeneration cycles) the total capacity of 1 ml beads is up to 50 μg of mRNA.

Recommended Buffers/Solutions

All common buffers for mRNA purification and isolation can be used with Dynabeads® Oligo (dT)25. To take full advantage of the unique properties of the beads, the buffers described below are recommended. All buffers should be brought to room temperature prior to use, apart from the 10 mM Tris-HCl which should be kept on ice o  at 2-8°C.

Binding Buffer:

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

Lysis/Binding Buffer:

100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA,
1% LiDS, 5 mM dithiothreitol (DTT). If any precipitation
is observed, warm to room temperature and
shake until all the components are fully resuspended.

Washing Buffer A:

10 mM Tris-HCl, pH 7.5 ,0.15 M LiCl, 1 mM EDTA, 0.1% LiDS

Washing Buffer B:

10 mM Tris-HCl, pH 7.5 ,0.15 M LiCl, 1 mM EDTA 10 mM Tris-HCl, pH 7.5

Reconditioning Solution:

0.1 M NaOH

Storage Buffer Oligo (dT)25:
250 mM Tris-HCl, pH 7.5, 20 mM EDTA, 0.1% Tween-20, 0.02% NaN3

Additional Material Needed

  • Magnetic device (Dynal® MPC™, Magnetic Particle Concentrator) for manual protocol. Dynal® MPC™-S is recommended for 20 μl – 2 ml samples (Cat. No. 120.20D)
  • Mixing/rotation device allowing both tilting and rotation
  • Sterile and RNase-free test tubes and pipette tips
  • Buffers/solutions
  • Water bath or heating block


For tissue samples:

  • Liquid nitrogen
  • Manual tissue grinder
  • Syringe and needle

All reagents used should be analytical grade and RNase-free.

Storage and Stability

When stored unopened at 2-8°C, this product is stable until the expiration date stated on the label. Do not freeze the product. Resuspend well before use. Store opened vials in an upright position and avoid bacterial contamination. Do not store the Dynabeads® in distilled water. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. If the beads dry out, resuspend them in the buffer in which they are supplied and leave to mix continuously overnight at 4°C. This treatment will completely restore their function. Dynabeads® Oligo (dT)25 are stable over a pH range of 4-13.


Warnings and Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. This product 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 buildup. Preservatives such as sodium azide are toxic if ingested.

Avoid pipetting by mouth.

The suspension of Dynabeads® Oligo (dT)25 is produced and quality controlled to be ribonuclease-free and thoroughly tested for optimal performance. Precautions should be taken to prevent RNase contamination of opened vials. Standard methods must be taken to prevent contamination by RNases during the preparation of mRNA. Follow appropriate laboratory guidelines.

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References

  1. Fellmann F et.al. Simplified Protocol of solid-phase cDNA libraries for multiple PCR amplification. BioTechniques 1996;21:766-770. (Ref.No. 1879)

  2. Jakobsen KS et al. Direct mRNA isolation using Magnetic Oligo(dT) Beads: A protocol for all types of cell cultures, animal and plant tissues. In Advances in Biomagnetic Separation, Ed. Uhlén, M., Hornes, E. and Olsvik, Ø., Eaton Publishing, 1994, pp. 61-71. (Ref. No. 1251)

  3. Karrer EE et al. In situ isolation of mRNA from individual plant cells: Creation of cell-specific cDNA libraries. Proc. Natl. Acad. Sci. USA 1995;92:3814- 3818. (Ref. No.1520)

  4. Lambert KN, Williamson VM. NA library construction from small amounts of RNA using paramagnetic beads and PCR. Nucleic Acids Res. 1993;21:775-776. (Ref. No. 911)

  5. Raineri I et al. Improved efficiency for singlesided PCR by creating a reusable pool of first strand cDNA coupled to a solid phase. Nucleic Acids Res. 1991;19:4010. (Ref. No. 540)

  6. Rodriguez IR, Chader GJ. A novel method for the isolation of tissue-specific genes. Nucleic Acids Res. 1992;20:3528. (Ref. No. 715)

  7. Stinear T et al. Detection of a Single Viable Cryptosporidium parvum Oocyst in Environmental Water Concentrates by Reverse Transcription-PCR. Applied and Environmental Microbiology 1996;62(9):3385-3390 (Ref. No. 1883)

  8. Aasheim HC et.al. Subtractive hybridization for the isolation of differentially expressed genes using magnetic beads. Methods in Molecular Biology 1996;69:115-128. (Ref. No. 1882)
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610.02_05_50.indd        Rev 006     5-May-2008