Related Product Information

Introduction

Kit Contents

Product number610.11610.12
Dynabeads® Oligo (dT)25*5 ml10 ml
Lysis/Binding Buffer30 ml60 ml
Washing Buffer A60 ml120 ml
Washing Buffer B30 ml60 ml
10 mM Tris-HCl (Elution Buffer)15 ml15 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

  • Magnets: See www.lifetechnologies.com/magnets for magnet recommendations.
  • RNase free pipette tips and pipettors.
  • RNase free microtubes.
  • Mixer allowing both tilting and rotation.
  • Heat block and/or incubator at 65-80°C for elution step (if required).

For isolation of mRNA from tissue samples:

  • Liquid nitrogen.
  • Mechanical or manual tissue grinder.
  • Syringe and 21 gauge needle.
  • Benchtop microcentrifuge.

Protocols

Technical Advice

  • Keep Dynabeads® Oligo (dT)25 in liquid suspension during storage and all handling steps. Resuspend well before use.
  • Work RNase free and wear gloves.
  • Bring all buffers, except the 10mM Tris-HCl (Elution Buffer), to room temperature prior to use. The 10mM Tris-HCl buffer should be stored on ice or at 2-8°C prior to use.
  • Thorough resuspension of the beads/mRNA complex during washing and complete removal of the washing buffer at each step will prevent carry over of LiDS and other salts to the downstream reaction. Transferring the beads/mRNA complex to new tubes before the last washing step will further reduce LiDS carry over. LiDS is a strong inhibitor of enzymatic reactions.


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

  1. Aliquot (weigh) the animal or plant tissue while frozen, to avoid mRNA degradation. Ideally the tissue should be weighed and aliquoted before freezing. Do not exceed the specified amount of tissue, as using too much tissue will reduce the mRNA yield and purity.

  2. Grind frozen tissue in liquid nitrogen. Work quickly.

  3. Transfer the frozen powder to the appropriate volume of Lysis/Binding Buffer and homogenize until complete lysis is obtained (approx. 1-2 min). A rapid lysis in the Lysis/Binding Buffer is critical for preventing mRNA degradation.

  4. A DNA-shear step is advised for samples containing over 500,000 cells. Force the lysate 3-5 times through  21 gauge needle using a 1-2 ml syringe to shear the DNA. The reduction in viscosity should be noticeable. Repeated shearing causes foaming of the lysate due to detergent in the buffer, however, this should not effect the mRNA yield. The foam can be reduced by a 30 second centrifugation. The lysate can be frozen and stored at –80°C for later use.

  5. Prepare Dynabeads® Oligo (dT)25 as described below, and subsequently proceed with the mRNA isolation in "Direct mRNA Isolation Protocol".

B) Preparation of Lysate from Cultured Cells or Cell Suspension

  1. Pellet cells by centrifugation (e.g. at 400 g for 8 minutes at 4°C) and wash the pellet by resuspending in phosphate-buffered saline (PBS). Pellet cells by centrifugation again. The cell pellet can be used immediately, or frozen in liquid nitrogen or at –80°C for later use.

  2. Add the appropriate volume of Lysis/Binding Buffer to either a frozen cell pellet or to a fresh cell pellet. Perform a repeated passage of the solution through a pipette tip to obtain complete lysis. The release of DNA during lysis results in a viscous solution which confirms complete lysis.

  3. A DNA-shear step is advised for samples containing over 500,000 cells. Force the lysate 3-5 times through a 21 gauge needle using a 1-2 ml syringe to shear the DNA. The reduction in viscosity should be noticeable. Repeated shearing causes foaming of the lysate due to detergent in the buffer, however, this should not effect the mRNA yield. The foam can be reduced by a 30 second centrifugation. The lysate can be frozen and stored at –80°C for later use.

  4. Prepare Dynabeads® Oligo (dT)25 as described below in section below, and subsequently proceed with the mRNA isolation.


Preparation of Dynabeads® Oligo (dT)25

  1. Resuspend Dynabeads® Oligo(dT)25 thoroughly before use.

  2. Transfer the desired volume of beads from the stock tube to a RNase-free 1.5 ml microcentrifuge tube and place the tube on a magnet (e.g. DynaMagTM-2).

  3. After 30 seconds (or when the suspension is clear), remove the supernatant.

  4. Remove the tube from the magnet and wash the beads by resuspending in an equivalent volume of fresh Lysis/Binding Buffer. Optional: For very small bead volumes (mini and micro isolations) use 50–100 μl wash volume to ease handling..

  5. Proceed to section below.


Direct mRNA Isolation Protocol

  1. Remove the Lysis/Binding Buffer from the pre-washed Dynabeads® Oligo(dT)25 by placing on the magnet for 30 seconds, or until the suspension is clear.

  2. Remove the microtube from the magnet and add the sample lysate                                                           

  3. Pipette to resuspend the beads completely in the sample lysate. Incubate with continuous mixing (rotating or roller mixer) for 3-5 min. at room temperature to allow the polyA tail of the mRNA to hybridize to the oligo(dT)25 on the beads. Increase the incubation time if the solution is viscous.

  4. Place the vial on the magnet for 2 min. and remove the supernatant. If the solution is noticeably viscous, increase the time to approx. 10 min.

  5. Wash the beads/mRNA complex two times with the appropriate volume of Washing Buffer A at room temperature. Use the magnet to separate the beads from the solution between each washing step.

  6. Wash the beads/mRNA complex once with the appropriate volume of Washing Buffer B at room temperature  Use the magnet to separate the beads from the solution.

  7. If the isolated mRNA is to be used in enzymatic downstream applications (e.g. RT-PCR), one extra wash in Washing Buffer B is recommended. This should be followed by a final wash in the enzymatic buffer to be used (e.g. RT-PCR buffer without the enzyme or primers).

    8. Note:       Perform cDNA synthesis as recommended by the manufacturer of the reverse transcriptase. When using a thermostable reverse transcriptase and the bead-bound oligo (dT) as primer for first strand cDNA synthesis, an initial incubation at 50°C for 5 minutes is necessary before proceeding at the recommended temperature.

  8. If elution of mRNA from the beads is desired, add an appropriate volume of 10 mM Tris-HCl (Elution Buffer) and incubate at 65-80°C for 2 min. Immediately place the tube on the magnet, transfer the supernatant containing the mRNA to a new RNase free tube and place this tube on ice.



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.

  1. Follow the isolation protocol (Direct mRNA Isolation Protocol above). Elute the mRNA in 10 mM Tris-HCl (Elution Buffer). Transfer the eluted mRNA to a new tube and place on ice. Do not discard the beads.

  2. Wash the beads two times in Washing Buffer B.

  3. Dilute the eluted mRNA in 4 times its volume of Lysis/Binding Buffer (e.g. if the mRNA is eluted in 20 μl, add 80 μl of Lysis/Binding Buffer.)

  4. Remove the Washing Buffer B from the beads, by placing the tube on the magnet, and add the diluted mRNA  Incubate with mixing at room temperature for 3-5 min.


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 cell10-30 pg
50 mg of muscle tissue50-80 μg
50 mg of liver tissue400 μg
107 cultured fibroblasts50-80 μg
107 cultured epithelial cells100-120 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 tissue100-400 mg20-100 mg4-20 mg≤ 4 mg
Animal tissue50-200 mg10-50 mg2-10 mg≤ 2 mg
Cultured cells4-20 × 1061-4 × 1060.15-1 × 106≤ 150,000
Dynabeads® Oligo(dT)251 ml250 μl50 μl10 μl
Lysis/Binding Buffer5 ml1250 μl300 μl300 μl
Washing Buffer A10 ml1-2 ml600 μl600 μl
Washing Buffer B5 ml1-1.5 ml300 μl300 μl
Tris-HCl (elution is optional)50-100 μl10-25 μl10 μl10 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 Rat4
BrainMouse, Trout12, 4
Brain (cereberal
cortex, preoptic area,
dentate gyrus)		Rat13, 14, 15
CartilageHuman16
Organ of Corti and
spiral ganglion		Guinea pig, rat17
Ear (cochleae)Mouse11
EggsTrout4
Gut
(paraffin embedded)		Human18
HeartRat13, 19*
HypothalamusRat15
KidneyRat13
Kidney (glomerular
preparations)		Human20
Liver (paraffin embedded)Human18
LiverRat, trout, Xenopus13, 19*, 4
Lung (paraffin embedded)Human18
LungRat13
MuscleRat, trout19*, 4
Nematode (frozen
rehydrated cysts)		Globodera
rostochiensis		58
OvariesTrout, Xenopus4
PancreasRat19*
Paraffin embedded
lung, liver, gut		Human18
Paraffin embedded
keratinocytes		Human21
PituitaryRat22
PlasmaHuman59
PronephosTrout4
Skin (dried)Frog57
SpleenRat4, 13
TrematodeSchistosoma mansoni23
Whole insectDrosophila4


*Lysis buffer with 4 M urea and 1 % SDS

Table 4.   mRNA DIRECT from plant tissues

Tissue Species References
Whole plantsArabidopsis thaliana
Rice, Oryza sativa		23, 24, 25, 26
27
BudTobacco28
Epidermal leaf cell
(single cells)		Tomato 1
EmbryosMaize, tobacco29
FlowersGuinea pig, rat17
Guard cell in leaf
(single cells)		Tomato1
LeavesBarley
Brassica oleracea
Maize
Potato
Tobacco
Tomato		31, 4, 32
33
29, 27
34
28
1
OvulesMaize29
RootsBarley
Brassica oleracea
Spruce
Maize		31
33
6
29
Seed aleuroneBarley31, 4, 35, 36, 32
Seed endospermBarley35, 36, 32
Seed embryosBarley31, 4, 35, 36, 32
SeedlingsMaize, tobacco29, 28
Single leaf cellsTomato 1
StemTobacco28
StigmaBrassica oleracea33, 37
Stolon tipsPotato34

 


Table 5.   mRNA DIRECT from different types of cells

Cell type/cell line  Origin  References
ChondrocytesHuman16
Cervical cancer cells
(HeLa)		Human38, 39
Colon carcinoma cell line
(COLO320)		Human40
Fibroblast cells line
(ST-1 and SKB-1)		Human41, 9
Fibroblast (D551)Human8, 9
Fibroblast (RTG-2)Trout4
Endothelial cells
(umbilical cord)		Human38, 9
Hepatocyte cell line
(HepG2)		Human8, 39
KeratinocytesHuman42, 21
Langerhans cellsHuman42
Lymphoblast B-cell
lines (Reh, Daudi,
HL-60, IM9)		Human8,4,39,43
Mamma carcinoma
cells (MCF7)		Human38, 39
Mamma carcinoma
(T47D)		Human40
MonocytesHuman44
Pancreas, insulinoma
Rinm5F cells		Rat45
Peripheral blood
mononuclear cells
(PBMC)		Human46
Peritoneal exudate
cells		Human42
Placental cell line
(AMA)		Human38, 39
T-cells/T-cell clonesHuman2, 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-147¹
Cerebrospinal fluidHIV-153¹
Cell lineHTLV-I/II54²
Peripheral blood
mononuclear cells
(PBMC)		HIV-147¹
PlasmaHIV-1/HIV-254¹ ²
SerumHIV-155¹, 56¹, 54¹ ²
T-lymphocytes cell line
(CD4+)		HIV-147¹


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.

Troubleshooting

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.

Comment

Using the appropriate bead-to-sample ratio (volume and concentration) there is no bias in hybridization based on mRNA size. However, with a large excess of mRNA or short incubation time, binding to the beads may be biased towards the short molecules. A similar bias may occur if the sample volumeto- bead ratio is too high.

General Information

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.

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