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Kit Contents
Product number | 610.11 | 610.12 |
---|---|---|
Dynabeads® Oligo (dT)25* | 5 ml | 10 ml |
Lysis/Binding Buffer | 30 ml | 60 ml |
Washing Buffer A | 60 ml | 120 ml |
Washing Buffer B | 30 ml | 60 ml |
10 mM Tris-HCl (Elution Buffer) | 15 ml | 15 ml |
*Approximately 5 mg/ml, supplied in PBS pH 7.4, containing 0.02% NaN3 as a preservative.
Product # 610.11 provides enough reagents for 20 standard isolations.
Product # 610.12 provides enough reagents for 40 standard isolations.
The suspension of Dynabeads® Oligo (dT)25 and the buffers provided are produced and packed under RNase-free conditions. All kit reagents are of analytical grade and are RNase-free.
Intended Use
This product has been designed for a simple and rapid isolation of pure, intact polyadenylated (polyA) mRNA directly from the crude lysate of animal and plant cells and tissues. The isolated mRNA is suitable for use in all downstream applications.
Principle
The isolation protocol relies on base pairing between the polyA 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 polyA tail will not hybridize to the beads and are readily washed away. RNase inhibiting agents in the Lysis/Binding Buffer together with stringent hybridization and washing conditions ensure the isolation of pure, intact mRNA from crude samples rich in RNase, without the use of strong chaotropic agents. The protocol is flexible and can easily be scaled up or down to suit all sample sizes. It has successfully been used in the isolation of mRNA from single cells. The high capture efficiency facilitates detection of mRNA by reverse transcriptase (RT)-PCR from highly specialized cells (e.g. isolated from a heterogeneous sample by immunomagnetic separation). In addition, the protocol has been successfully used to isolate mRNA from a wide variety of tissues of mammalian, fish, amphibian, insect and plant origins.
For many applications elution of the mRNA from the beads is not required as the beads do not interfere with downstream enzymatic reactions. The bead-bound oligo (dT)25 can also function as a primer for RT and synthesis of first-strand cDNA, allowing the construction of solid-phase cDNA libraries and solid-phase RT-PCR.
Binding Capacity
1 mg of beads (200 μl) will bind up to 2 μg of mRNA. A typical mammalian cell contains about 10-30 pg of total RNA, from which 1-5 % is mRNA.
Description of Materials
Characteristics of Dynabeads® Oligo (dT)25
Dynabeads® are uniform, superparamagnetic beads. They are stable in the pH range of 4-13. Do not freeze the 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: 120 ± 25 × 10-6 m3/kg
Buffers
Lysis/Binding Buffer
100 mM Tris-HCl, pH 7.5
500 mM LiCl
10 mM EDTA, pH 8
1% LiDS
5 mM dithiothreitol (DTT)
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 (Elution Buffer)
10 mM Tris-HCl, pH 7.5
Please note that precipitate may form in the buffers. Dissolve precipitate before use by warming to room temperature and mixing thoroughly.
Additional Material Required
Technical Advice
Protocols included
Preparation of Sample Lysate
The mRNA content of cells and tissues varies greatly depending on the source of the material and RNA expression levels at the time of tissue/ cell harvest. Dynabeads® mRNA DIRECT™ Kit protocols can be scaled up or down to suit specific sample source and quantity. Please see section "Sample Guidelines and Scaling" before preparing the sample, and for recommended bead and buffer volumes (Table 1 and 2).
A) Preparation of Lysate from Solid Plant or Animal Tissue
B) Preparation of Lysate from Cultured Cells or Cell Suspension
Preparation of Dynabeads® Oligo (dT)25
Direct mRNA Isolation Protocol
Re-use of Dynabeads® Oligo (dT)25 for Large Scale Isolations
Please note that the buffers supplied with the kit (product no. 610.11 and 610.12) may not be sufficient for large scale mRNA isolations. Multiple isolations from the same sample can be performed by re-using Dynabeads® Oligo(dT)25 after mRNA elution. Simply follow the protocol described in section "Direct mRNA Isolation Protocol". After elution of the mRNA, wash the beads once in Lysis/Binding Buffer (section above). Add a new lysate sample to the beads and continue the isolation as usual. Alternatively, washed beads can be re-applied to the same sample lysate until all the mRNA has been captured.
Elimination of rRNA Contamination
In some cases trace amounts of ribosomal RNA have been observed in the mRNA samples. For many applications such as Northern blotting and RT-PCR, trace amounts of rRNA contamination will not interfere with the analysis or interpretation of the results. However, for other applications such as cDNA library construction and microarray analysis, rRNA contamination should be avoided. Ribosomal RNA is effectively eliminated by re-extracting the mRNA from the eluate. Re-use of the same Dynabeads® Oligo(dT)25 used for the original isolation is recommended. If new beads are used, it is recommended that the beads are washed in 50 mM Sodiumpyrophosphate before the isolation of mRNA.
Continue with the isolation protocol ( Direct mRNA Isolation Protocol, starting at step 4.)
Sample Guidelines and Scaling
The following information is intended as a rough guide to the expected total RNA content of selected tissues, as well as appropriate bead and buffer volumes.
Table 1. Estimated total RNA yield from mammalian cells and tissues
Cell Types & Quantity | Estimated Total RNA Content (1-5% is mRNA) |
---|---|
Single mammalian cell | 10-30 pg |
50 mg of muscle tissue | 50-80 μg |
50 mg of liver tissue | 400 μg |
107 cultured fibroblasts | 50-80 μg |
107 cultured epithelial cells | 100-120 μg |
Table 2. Recommended volumes of Dynabeads® Oligo(dT)25 and buffers for use with different amounts of starting material
Components | Maxi | Standard | Mini | Micro |
---|---|---|---|---|
Plant tissue | 100-400 mg | 20-100 mg | 4-20 mg | ≤ 4 mg |
Animal tissue | 50-200 mg | 10-50 mg | 2-10 mg | ≤ 2 mg |
Cultured cells | 4-20 × 106 | 1-4 × 106 | 0.15-1 × 106 | ≤ 150,000 |
Dynabeads® Oligo(dT)25 | 1 ml | 250 μl | 50 μl | 10 μl |
Lysis/Binding Buffer | 5 ml | 1250 μl | 300 μl | 300 μl |
Washing Buffer A | 10 ml | 1-2 ml | 600 μl | 600 μl |
Washing Buffer B | 5 ml | 1-1.5 ml | 300 μl | 300 μl |
Tris-HCl (elution is optional) | 50-100 μl | 10-25 μl | 10 μl | 10 μl |
Sample Types from which mRNA has been Isolated Using Dynabeads® Oligo (dT)25
Table 3. mRNA DIRECT from animal tissues
Tissue | Species | References |
---|---|---|
Adrenals | Rat | 4 |
Brain | Mouse, Trout | 12, 4 |
Brain (cereberal cortex, preoptic area, dentate gyrus) | Rat | 13, 14, 15 |
Cartilage | Human | 16 |
Organ of Corti and spiral ganglion | Guinea pig, rat | 17 |
Ear (cochleae) | Mouse | 11 |
Eggs | Trout | 4 |
Gut (paraffin embedded) | Human | 18 |
Heart | Rat | 13, 19* |
Hypothalamus | Rat | 15 |
Kidney | Rat | 13 |
Kidney (glomerular preparations) | Human | 20 |
Liver (paraffin embedded) | Human | 18 |
Liver | Rat, trout, Xenopus | 13, 19*, 4 |
Lung (paraffin embedded) | Human | 18 |
Lung | Rat | 13 |
Muscle | Rat, trout | 19*, 4 |
Nematode (frozen rehydrated cysts) | Globodera rostochiensis | 58 |
Ovaries | Trout, Xenopus | 4 |
Pancreas | Rat | 19* |
Paraffin embedded lung, liver, gut | Human | 18 |
Paraffin embedded keratinocytes | Human | 21 |
Pituitary | Rat | 22 |
Plasma | Human | 59 |
Pronephos | Trout | 4 |
Skin (dried) | Frog | 57 |
Spleen | Rat | 4, 13 |
Trematode | Schistosoma mansoni | 23 |
Whole insect | Drosophila | 4 |
*Lysis buffer with 4 M urea and 1 % SDS
Table 4. mRNA DIRECT from plant tissues
Tissue | Species | References |
---|---|---|
Whole plants | Arabidopsis thaliana Rice, Oryza sativa | 23, 24, 25, 26 27 |
Bud | Tobacco | 28 |
Epidermal leaf cell (single cells) | Tomato | 1 |
Embryos | Maize, tobacco | 29 |
Flowers | Guinea pig, rat | 17 |
Guard cell in leaf (single cells) | Tomato | 1 |
Leaves | Barley Brassica oleracea Maize Potato Tobacco Tomato | 31, 4, 32 33 29, 27 34 28 1 |
Ovules | Maize | 29 |
Roots | Barley Brassica oleracea Spruce Maize | 31 33 6 29 |
Seed aleurone | Barley | 31, 4, 35, 36, 32 |
Seed endosperm | Barley | 35, 36, 32 |
Seed embryos | Barley | 31, 4, 35, 36, 32 |
Seedlings | Maize, tobacco | 29, 28 |
Single leaf cells | Tomato | 1 |
Stem | Tobacco | 28 |
Stigma | Brassica oleracea | 33, 37 |
Stolon tips | Potato | 34 |
Table 5. mRNA DIRECT from different types of cells
Cell type/cell line | Origin | References |
---|---|---|
Chondrocytes | Human | 16 |
Cervical cancer cells (HeLa) | Human | 38, 39 |
Colon carcinoma cell line (COLO320) | Human | 40 |
Fibroblast cells line (ST-1 and SKB-1) | Human | 41, 9 |
Fibroblast (D551) | Human | 8, 9 |
Fibroblast (RTG-2) | Trout | 4 |
Endothelial cells (umbilical cord) | Human | 38, 9 |
Hepatocyte cell line (HepG2) | Human | 8, 39 |
Keratinocytes | Human | 42, 21 |
Langerhans cells | Human | 42 |
Lymphoblast B-cell lines (Reh, Daudi, HL-60, IM9) | Human | 8,4,39,43 |
Mamma carcinoma cells (MCF7) | Human | 38, 39 |
Mamma carcinoma (T47D) | Human | 40 |
Monocytes | Human | 44 |
Pancreas, insulinoma Rinm5F cells | Rat | 45 |
Peripheral blood mononuclear cells (PBMC) | Human | 46 |
Peritoneal exudate cells | Human | 42 |
Placental cell line (AMA) | Human | 38, 39 |
T-cells/T-cell clones | Human | 2, 47, 48, 49, 50 |
Yeast (Saccharomyces cerevisiae, Hansenula polymorpha) | In soil samples | 51 |
Yeast (Saccharomyces cerevisiae) | Culture | 52 |
Table 6. Direct isolation of viral polyA RNA with Dynabeads® Oligo (dT)25
Starting Material | Virus | References |
---|---|---|
Cells in bronchoalveolar washes | HIV-1 | 47¹ |
Cerebrospinal fluid | HIV-1 | 53¹ |
Cell line | HTLV-I/II | 54² |
Peripheral blood mononuclear cells (PBMC) | HIV-1 | 47¹ |
Plasma | HIV-1/HIV-2 | 54¹ ² |
Serum | HIV-1 | 55¹, 56¹, 54¹ ² |
T-lymphocytes cell line (CD4+) | HIV-1 | 47¹ |
1) Lysis/binding buffer: 1 M LiCl, 2% SDS, 2xTE, 50 μg tRNA, Vanadyl ribonucleosyl complexes.
2) Lysis/binding buffer: 4 M GTC, 0.5% sarkosyl, 1% DTT, 0.5 M LiCl, 0.1 M Tris pH8.
Problem | Possible Cause | Suggested Solution |
---|---|---|
Clumping of beads during incubation step with sample lysate. | DNA in the sample lysate has not been completely sheared. | i) Pipette the solution several times through a 1 ml pipette. ii) Increase force/number of passages through the needle in future shearing steps. |
mRNA is contaminated with DNA. | i) Incomplete DNA shearing. ii) Incomplete removal of sample lysate after hybridization step, and subsequent carry over to wash and elution steps. iii) Inefficient washing. iv) Incomplete removal of wash buffers. v) Sample-to-beads ratio too high. | i) Increase the force and /or the number of passages through the needle in the DNA shearing step. ii) Completely remove the sample lysate after hybridization. iii) Make sure the beads/mRNA complex is fully resuspended in washing buffer. iv) Completely remove the sample/washing buffers. v) Dilute sample lysate or increase the amount of beads. vi) Re-extract the mRNA from the eluate. |
mRNA yield is lower than expected. | i) Inefficient elution of mRNA from the beads. ii) Beads-to-sample ratio is too low. iii) Cells/tissue incompletely lysed. | i) Increase the elution volume/time/temperature or perform the elution step two times, pooling the eluate. ii) Increase the amount of beads. iii) Repeat the homogenization step. |
The beads/cDNA complex is clumped and sticking to the tubes after reverse transcription. | Non-specific electrostatic interactions between the cDNA molecules and the plastic materials of the tubes/pipette tips. | i) Add BSA (0.2-1.0% final concentration) to the reverse transcription mix before performing the cDNA synthesis. This is to reduce clumping of the beads for a more efficient cDNA synthesis. Note: use best possible BSA quality. ii) Where appropriate, add 0.05% Tween-20 to the reaction buffers. iii) Alternatively, dilute the beads/cDNA solution (after reverse transcription) with an equal volume of the 1 × reverse transcription reaction buffer containing 0.05% Tween-20. Mix by pipetting and transfer the suspension to a new tube. If there are any remaining beads stuck to the tube walls, remove by washing with a fresh aliquot of buffer containing Tween-20. Pool these beads with the original bead suspension. Place the pooled beads on a magnet and remove the supernatant, then wash 2-3 times with the buffer containing Tween-20. Store the solid-phase cDNA library in an appropriate buffer containing 0.05% Tween-20. |
Unable to detect specific mRNA molecules. | i) The beads-to-sample ratio is too low. ii) Inappropriate sample volume. iii) Hybridization time too short. | i) Increase the amount of beads. ii) Reduce sample volume/increase sample concentration. iii) Increase hybridization incubation time to 10-15 min. |
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. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.
Technical Support
Please contact Invitrogen Dynal® for further technical support (see contact details). Certificate of
Analysis/Compliance is available upon request.
Warning and Limitations
This product is for research use only. The product is not for use in human diagnostics or therapeutic procedures. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide (NaN3) as a preservative, which is cytotoxic. Avoid pipetting by mouth!
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. Material Safety Data Sheet (MSDS) is available at http://www.lifetechnologies.com.