Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
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.
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.
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 cultured cells and cell suspensions
Isolation of mRNA from crude lysate
Purification of mRNA from total RNA
In the below example, mRNA is purified from 75 μg of total RNA starting material.
Regeneration and reuse of Dynabeads® Oligo (dT)25
The oligo (dT) sequences are covalently attached to the surface of the Dynabeads® . This enables single stephybridization, 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.
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
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.
Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.
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
For tissue samples:
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.