Introduction

System Overview

The FastTrack® MAG mRNA Isolation Kit uses oligo(dT)-conjugated magnetic beads to isolate poly A+ RNA directly from cell lysate, tissue lysate, or total RNA in 1–1.5 hours using conventional laboratory equipment (Morrissey et al., 1989; Stone et al., 1996). The high quality and quantity of the isolated mRNA makes it suitable for use in a variety of applications, including cDNA library construction, microinjection, in vitro translation, RT-PCR, Northern blotting. The mRNA captured on the magnetic beads may also be used in some applications without elution. Using the kit, you can isolate mRNA from varying amounts of cells (<1 × 105 – 5 × 107 cells), tissue samples (< 10 mg–> 300 mg of tissue), or total RNA (< 1 μg–2000 μg). Separate procedures are provided for each type of sample.

Workflow

Using the FastTrack® MAG mRNA Isolation Kit, you first isolate total RNA using a method of choice, or prepare cells/tissue samples using the reagents provided in this kit. Then you prewash the FastTrack® MAG Beads, bind the sample, and perform a series of wash steps. Finally, you elute the mRNA from the beads using RNase-free water provided in the kit.

Isolate Total RNA           Lyse Cells        Homogenize tissue                20-30 Minutes
         
     ↓                                     ↓                            ↓

         Prewash FastTrack® MAG Beads and bind sample                    20-30 Minutes                 
                                                   
  ↓
                              
                               Wash FastTrack® MAG Beads                                    10-20 Minutes
                                                      ↓
                  
                                              Elute sample                                                    5-10 Minutes



Advantages of the Kit

The FastTrack® MAG mRNA Isolation Kit offers the following advantages:

  • Isolates high-quality mRNA in less than 1 hour from total RNA and 1.5 hours from cells or tissue
  • Isolated mRNA has minimal ribosomal RNA and genomic DNA contamination
  • Higher yields of mRNA than comparable systems
  • mRNA may be eluted at a high concentration for specific downstream applications
  • Compatible with high-throughput applications

Typical Yields

Source Recovery of mRNA
1 mg total RNA (from mammals)>30 μg (>3%)
1 mg total RNA (from plants)>15 μg (>1.5%)
1 × 106 cells1 μg
1 g tissue5–80 μg*

*Yields of mRNA from tissue are highly dependent on the type of tissue.

Magnetic Particle Separator

This kit requires the use of a magnetic particle separator (MPS) with holes for 1.5-ml tubes to separate the FastTrack® MAG beads in solution. The Magna-Sep™ MPS is a 6-hole magnetic particle separator available from Invitrogen that can hold 1.5–2.0-ml tubes (Catalog no. K1585-01). You may also use separators from other manufacturers.
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Ordering Information

Experienced Users Procedure to Isolate mRNA from Total RNA

Introduction

This quick reference sheet is included for experienced users of the FastTrack® MAG mRNA Isolation Kits. If you are a first time user, refer to the details in the manual.


Use the listed amount of reagents with the specified amount of  total RNA for the purification procedure described below.



Amount Micro   Maxi  
Total RNA< 1 μg1–50 μg50–500 μg500–2000 μg
FastTrack® MAG Beads20 μl50 μl100 μl≥ 200 μl
Binding Buffer B6100 μl200 μl500 μl500 μl
Wash Buffer W7100 μl × 6200 μl × 6500 μl × 6500 μl × 6




Step - Total RNA Protocol                                                        Procedure




  1. Isolate total RNA. Perform DNase I digestion, if you wish to use the mRNA for RT-PCR.

  2. Preheat the specified volume of Binding Buffer B6 (see above table) at 65-70°C.

  3. Add RNase-free water to your total RNA sample to a total volume equal to the volume of Binding Buffer B6 in Step 2. Place the sample tube on ice until use.

  4. Gently pipet the FastTrack® MAG Beads up and down to thoroughly resuspend the beads. Transfer the specified amount of resuspended beads (see above table) to an RNase-free microcentrifuge tube.

  5. Insert the tube into the magnetic particle separator (MPS) for ∼0.5–2 minutes. Remove and discard the supernatant.

  6. Immediately add the specified volume of Wash Buffer W7.

  7. Remove the tube from the MPS and resuspend the beads by pipetting gently up and down.

  8. Repeat Steps 5-6 one more time.

  9. Insert the tube into the MPS for ∼0.5–2 minutes. Remove and discard the supernatant.

  10. Immediately add the total RNA sample from Step 3, and the heated Binding Buffer B6 from Step 2, above.

  11. Remove the tube from the MPS and resuspend beads by pipetting gently up and down.

  12. Incubate the capped tube at 65–70°C for 2–5 minutes.

  13. Transfer the tube to a rotator and rotate the sample for 10 minutes at room temperature

  14. Insert the tube in the MPS for ∼0.5–2 minutes. Remove and save the supernatant.

  15. Immediately add the specified volume of Wash Buffer W7.

  16. Remove the tube from the MPS and resuspend the beads by pipetting gently up and down.

  17. Reinsert the tube in the MPS for ∼0.5–2 minutes. Remove and discard the wash buffer from the tube.

  18. Repeat Steps 15-17 three more times.

  19. Immediately add 5-20 μl RNase-free water for <50 μg total RNA starting amount or add 20–50 μl RNase-free water for >50 μg total RNA starting amount.

  20. Remove the tube from the MPS and resuspend beads by pipetting. Incubate at 37°C for 2–5 minutes.

  21. Insert the tube into the MPS for∼0.5–2 minutes. Remove and save the supernatant.
    Important: The supernatant contains the isolated mRNA.

  22. Repeat Steps 19-21 using 5 μl RNase-free water for <50 μg total RNA and 10 μl RNase-free water for >50 μg total RNA. Again, save the supernatant.

  23. Combine the supernatants from Steps 21 and 22 containing your isolated mRNA.

  24. Store mRNA at -80ºC.

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Experienced Users Procedure to Isolate mRNA from Cells

Introduction

This quick reference sheet is included for experienced users of the FastTrack® MAG mRNA Isolation Kits. If you are a first time user, refer to the details in regular procedures below.


Use the listed amount of reagents with the specified amount of cells for the purification procedure described below



Amount Micro   Maxi  
Cells<1 × 105105–106106–107107–5 × 107
FastTrack® MAG Beads20 μl20-50 μl50-100 μl100-200 μl
Binding Buffer B6100 μl200 μl500 μl500 μl
Wash Buffer W6
100 μl200 μl500 μl500 μl
Wash Buffer W7100 μl × 5200 μl × 5500 μl × 5500 μl × 5




Step - Cells Protocol                                                        Procedure




  1. Harvest cells and prepare cell lysate.

  2. Preheat the specified volume of Binding Buffer B6 (see above table) at 65-70°C.

  3. Gently pipet the FastTrack® MAG Beads up and down to thoroughly resuspend the beads. Transfer the specified amount of resuspended beads (see above table) to an RNase-free microcentrifuge tube.

  4. Insert the tube into the MPS for ∼0.5–2 minutes. Remove and discard the supernatant.

  5. Immediately add the specified volume of Wash Buffer W7.

  6. Remove the tube from the MPS and resuspend the beads by pipetting gently up and down.

  7. Repeat Steps 4-6 one more time.

  8. Insert the tube into the magnetic particle separator (MPS) for ∼0.5–2 minutes. Remove and discard the supernatant.

  9. Immediately add the cell lysate, and the heated Binding Buffer B6 from Step 2, above.

  10. Remove the tube from MPS and resuspend the beads by pipetting up and down.

  11. Incubate the capped tube at 65–70°C for 2–5 minutes.

  12. Transfer the tube to a rotator and rotate the sample for 10 minutes at room temperature.

  13. Insert the tube in the MPS for ∼2–5 minutes. Remove and save the supernatant.

  14. Immediately add the specified volume of Wash Buffer W6.

  15. Remove the tube from the MPS and resuspend the beads by pipetting.

  16. For >1 × 106 cells and/or performing downstream RT-PCR: Add 1 μl (1 unit) of DNase I, Amplification Grade, per 100 μl Wash Buffer W6 to the tube. Mix by pipetting gently up and down and incubate at 25°C for 5–10 minutes.

  17. Insert the tube in the MPS for ∼0.5–2 minutes. Remove and discard the wash buffer from the tube.

  18. Immediately add the specified volume of Wash Buffer W7.

  19. Remove the tube from the MPS and resuspend the beads by pipetting gently up and down.

  20. Insert the tube in the MPS for ∼0.5–2 minutes. Remove and discard the wash buffer.

  21. Repeat Steps 18-20 using Wash Buffer W7 two more times.

  22. Immediately add 5-20 μl RNase-free water for a starting amount of <1 × 106 cells or add 20–50 μl RNase-free water for a starting amount of >1 × 106 cells.

  23. Remove the tube from the MPS and resuspend the beads by pipetting. Incubate at 37°C for 2–5 minutes.

  24. Insert the tube into the MPS for ∼0.5-2 minutes. Remove and save the supernatant. Important: The supernatant contains the isolated mRNA.

  25. Repeat Steps 22-24, using 5 μl RNase-free water for <1 × 106 cells and 10 μl RNase-free water for >1 × 106 cells.

  26. Combine the supernatants from Steps 24 and 25 containing your isolated mRNA. Store mRNA at -80°C.

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Experienced Users Procedure to Isolate mRNA from Tissues

Introduction

This quick reference sheet is included for experienced users of the FastTrack® MAG mRNA Isolation Kits. If you are a first time user, refer to the details in regular procedures below.


Use the listed amount of reagents with the specified amount of tissue for the purification procedure described below



Amount Micro   Maxi  
Tissue< 10 mg10–50 mg50–200 mg200–500 mg
FastTrack® MAG Beads20 μl20-50 μl50-100 μl100-200 μl
Binding Buffer B6100 μl200 μl500 μl500 μl
Wash Buffer W6
100 μl200 μl500 μl500 μl
Wash Buffer W7100 μl × 5200 μl × 5500 μl × 5500 μl × 5




Step - Tissue Protocol                                                        Procedure




  1. Harvest tissue and prepare tissue lysate.

  2. Preheat the specified volume of Binding Buffer B6 (see above table) at 65-70°C.

  3. Gently pipet the FastTrack® MAG Beads up and down to thoroughly resuspend the beads. Transfer the specified amount of resuspended beads (see above table) to an RNase-free microcentrifuge tube.

  4. Insert the tube into the MPS for ∼0.5–2 minutes. Remove and discard the supernatant.

  5. Immediately add the specified volume of Wash Buffer W7.

  6. Remove the tube from the magnetic particle separator (MPS) and resuspend the beads by pipetting gently up and down.

  7. Repeat Steps 4-6 one more time with Wash Buffer W7.

  8. Insert the tube into the MPS for ∼0.5–2 minutes. Remove and discard the supernatant.

  9. Immediately add the tissue lysate, and the heated Binding Buffer B6 from Step 2, above.

  10. Remove the tube from MPS and resuspend the beads by pipetting up and down.

  11. Incubate the capped tube at 65–70°C for 2–5 minutes.

  12. Transfer the tube to a rotator and rotate the sample for 10 minutes at room temperature.

  13. Insert the tube in the MPS for ∼2–5 minutes. Remove and save the supernatant.

  14. Immediately add the specified volume of Wash Buffer W6.

  15. Remove the tube from the MPS and resuspend the beads by pipetting gently up and down.

  16. For >50 mg tissue and/or performing downstream RT-PCR: Add 1 μl (1 unit) DNase I, Amplification Grade, per 100 μl Wash Buffer W6 to the tube. Mix by pipetting and incubate at 25°C for 5–10 minutes.

  17. Insert the tube in the MPS for∼0.5–2 minutes. Remove and discard the wash buffer from the tube.
  18. Immediately add the specified volume of Wash Buffer W7.

  19. Remove the tube from the MPS and resuspend the beads by pipetting gently up and down.

  20. Insert the tube in the MPS for ∼0.5–2 minutes. Remove and discard the wash buffer from the tube.

  21. Repeat Steps 18-20 using Wash Buffer W7 two more times.

  22. Immediately add 5–20 μl RNase-free water for <50 mg tissue starting material or add 20–50 μl RNase-free water for >50 mg of tissue starting material.

  23. Remove the tube from the MPS and resuspend the beads by pipetting. Incubate at 37°C for 2–5 minutes.

  24. Insert the tube into the MPS for ∼0.5–2 minutes. Remove and save the supernatant. Important: The supernatant contains the isolated mRNA.

  25. Repeat Steps 22-24, using 5 μl RNase-free water for <50 mg of tissue and 10 μl RNase-free water for >50 mg of tissue. Again, save the supernatant.

  26. Combine the supernatants from Steps 24 and 25 containing your isolated mRNA. Store mRNA at -80°C.

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Handling RNA

General Handling of RNA

When working with RNA:

  • Do not use plasticware or glassware without first eliminating possible ribonuclease contamination (see below).
  • Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin.
  • Always use proper microbiological aseptic technique when working with RNA.

RNase AWAY® Reagent

We recommend RNase AWAY® Reagent for removing RNase and DNA contamination from surfaces. This reagent is nonabrasive, noncarcinogenic, and nonbiologically corrosive.

RNase-free Plasticware

  • Use disposable, individually wrapped, sterile plasticware.
  • Use only sterile, new pipette tips and microcentrifuge tubes.
  • All plasticware provided in the FastTrack® MAG mRNA Isolation Kit is RNase-free.
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Isolating mRNA from Total RNA

Introduction

This section provides procedures for isolating mRNA from total RNA.

User Supplied Materials

  • Total RNA purification reagent/system
  • Magnetic particle separator (the Magna-Sep™ 6-hole MPS is available from Invitrogen
  • Heat block or thermal cycler preheated to 65–70°C
  • RNase-free 1.5-ml tubes
  • Pipettes
  • Rotator
  • Tabletop microcentrifuge

Isolating Total RNA

To isolate total RNA, we recommend TRIzol® Reagent (Cat. Nos. 15596-026/-018), the PureLink™ Micro-to-Midi Total RNA Purification System (Cat. no. 12183-018), or the PureLink™ 96 RNA Purification Kit (Cat. no. 12173-011).

Note:  If the total RNA contains high-level of genomic DNA or you are performing downstream RT-PCR, perform a DNase I digestion step to minimize genomic DNA contamination prior to mRNA purification.

Amounts of Beads and Buffers

The following table lists the amounts of FastTrack® MAG Beads, Binding Buffer B6, and Wash Buffer W7 to use with the specified amount of total RNA. Note: The isolation procedure uses six separate volumes of Wash Buffer W7, so each volume is shown × 6.

Amount Micro   Maxi  
Total RNA< 1 μg1–50 μg50–500 μg500–2000 μg
FastTrack® MAG Beads20 μl50 μl100 μl≥ 200 μl*
Binding Buffer B6100 μl200 μl500 μl500 μl
Wash Buffer W7100 μl × 6200 μl × 6500 μl × 6500 μl × 6

*To ensure maximum mRNA yield, the ratio of total RNA (μg) to beads (μl) should be > 7.5 (i.e., at least 200 μl of beads per 1500 μg of total RNA).

Before Proceeding

Before proceeding with the protocol, preheat the Binding Buffer B6 and add water to the total RNA sample as described below. While the buffer is heating, proceed with the Prewash and Binding Procedure above.

  • Add the volume of Binding Buffer B6 specified in the table on the previous page to a 1.5-ml RNase-free tube. Place the tube in a heat block or thermal cycler at 65-70°C.
  • In a separate 1.5-ml RNase-free tube, add RNase-free water to your total RNA sample to a total volume equal the volume of Binding Buffer B6 in Step 1. Place the sample tube on ice.

Important: 
When following the procedures in this section, be careful to never let the beads dry out.

Pipetting Liquid from Tubes

When pipetting liquid from a tube in the magnetic separator, always point the pipette tip toward the opposite side of the tube bottom from the pellet to avoid touching the beads.

Prewash and Binding Procedure

While the Binding Buffer B6 is preheating (see previous page), proceed with the steps below:

  1. Thoroughly resuspend the FastTrack® MAG Beads by pipetting them gently up and down. Transfer the amount of resuspended beads specified in the table on page 4 to an RNase-free microcentrifuge tube.

  2. Insert the microcentrifuge tube into the magnetic particle separator (MPS). When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the volume of Wash Buffer W7 specified in the table on page 4. Important: Do not let the beads dry out.

  3. Remove the tube from the magnetic particle separator and resuspend the beads by pipetting gently up and down.

  4. Repeat Steps 2–3 one more time, and then proceed to Step 5.

  5. Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the total RNA sample from Step 2 above and the heated Binding Buffer B6 from Step 1 above.

  6. Remove the tube from the magnetic separator and resuspend the beads in the solution by pipetting gently up and down. (Note: Pipetting too vigorously may damage the RNA.)

  7. Cap the tube and place in the heat block or thermal cycler. Incubate at 65–70°C for 2–5 minutes.

  8. Transfer the tube to a rotator and rotate the sample for 10 minutes at room temperature. Proceed to Wash and Elution Procedure below.

Wash and Elution Procedure

  1. Insert the tube from Prewash and Binding Procedure, Step 8, in the magnetic separator. When the beads are clearly separated (∼0.5–2 minutes), remove and save the supernatant. Note: For rare samples, if your yield is low, you may want to preserve this supernatant and attempt to perform a separate isolation with it.

  2. To the beads, immediately add the volume of Wash Buffer W7 specified in the table

  3. Remove the tube from the magnetic separator and resuspend the beads in the wash buffer by pipetting gently up and down.

  4. Reinsert the tube in the magnetic separator. When the beads are clearly separated (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Be careful to completely remove the wash buffer.

  5. Repeat Steps 2–4 three more times, and proceed to Step 6.

  6. If you started with < 50 μg of total RNA, immediately add 5-20 μl of RNase-free water. If you started with > 50 μg of total RNA, immediately add 20–50 μl of RNase-free water.

  7. Remove the tube from the magnetic separator and thoroughly resuspend the beads by pipetting gently up and down. Incubate at 37°C for 2–5 minutes. Use the longer incubation time for larger samples.

  8. Insert the tube into the magnetic separator. When the beads are clearly separated (∼0.5–2 minutes), remove and save the supernatant. Important: The supernatant contains the isolated mRNA. Do not discard.

  9. Repeat Steps 6–8, using 5 μl of RNase-free water for < 50 μg of total RNA and 10 μl of RNase-free water for > 50 μg of total RNA. Again, save the supernatant.

  10. Combine the supernatants from Steps 8 and 9. This is your isolated mRNA. Store mRNA at -80°C.

The mRNA quality and yield may be determined by spectrophotometry and agarose gel electrophoresis, as described above.
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Isolating mRNA from Cells

Introduction

This section provides procedures for isolating mRNA from cells.

User Supplied Materials

  • Magnetic particle separator (the Magna-Sep™ 6-hole MPS is available from Invitrogen
  • Heat block or thermal cycler preheated to 65–70°C
  • Water bath or incubator at 45°C
  • RNase-free 1.5-ml tubes
  • Pipettes
  • Rotator
  • 21-gauge needle and 1-ml syringe
  • Tabletop microcentrifuge
  • Optional for Micro kit: DNase I, Amplification Grade (Catalog no. 18068-015)

Amounts of Beads and Buffers

The following table lists the amounts of FastTrack® MAG Beads, Lysis Buffer, Proteinase K, Binding Buffer B6, and Wash Buffer W6, and Wash Buffer W7 to use with the specified amount of cells.

Note: The isolation procedure uses five separate volumes of Wash Buffer W7, so each volume is shown × 5.

Amount Micro   Maxi  
Cells< 1 × 1051 × 105–1061 × 106–1071 × 107– 5 × 107
FastTrack® MAG Beads20 μl20-50 μl50-100 μl100-200 μl
Lysis Buffer L4
100 μl
200 μl
500 μl
500 μl
Proteinase K
2.5 μl
5 μl
10 μl
10 μl
Binding Buffer B6100 μl200 μl500 μl500 μl
Wash Buffer W6
100 μl200 μl500 μl500 μl
Wash Buffer W7100 μl × 5200 μl × 5500 μl × 5500 μl × 5

*For cells amounts > 5 × 107, you can use more Lysis Buffer L4/Proteinase K and Binding Buffer B6 in multiple tubes or proportionally scale up to a larger tube.


Preparing Binding Buffer and Lysis Buffer

Preheat the Binding Buffer B6 and prepare the Lysis Buffer L4 as described below:

  1. Add the volume of Binding Buffer B6 specified in the table above to a 1.5-ml RNase-free tube. Place the tube in a heat block or thermal cycler at 65–70°C.

  2. In a separate 1.5-ml RNase-free tube, add the volume of Proteinase K specified in the table above to the specified volume of Lysis Buffer L4. Proceed with Cell Lysis, below.

Preparing Cells

Collect, wash, and pellet cells according to your standard laboratory protocol.

Cell Lysis

Lyse cells as described below.

  1. To the cell pellet in a microcentrifuge tube, add the Lysis Buffer solution from Step 2, above.

  2. For <1 × 105 cells, pipette the lysate up and down at least 10 times. For >1 × 105 cells, shear the DNA by passing the lysate through a 21-gauge needle fitted to a 1-ml syringe. Pass the lysate through the needle ≥20 times for Micro kit volumes and ≥30 times for Maxi kit volumes.

  3. Transfer the tube to a microcentrifuge and spin at maximum speed for 5 minutes at room temperature.

  4. Carefully transfer the supernatant containing your sample to a fresh microcentrifuge tube.

  5. Incubate the sample at 45°C in a water bath or incubator for 10 minutes. While the sample is incubating, proceed to Prewash and Binding Procedure below.

Pipetting Liquid from Tubes

When pipetting liquid from a tube in the magnetic separator, always point the pipette tip toward the opposite side of the tube bottom from the pellet to avoid touching the beads.

Important:  When following the procedures in this section, be careful to never let the beads dry out.


Prewash and Binding Procedure

While the lysate is incubating (above), proceed with the steps below:

  1. Thoroughly resuspend the FastTrack® MAG Beads by pipetting them gently up and down. Transfer the amount of resuspended beads specified in the table above to an RNase-free microcentrifuge tube.

  2. Insert the microcentrifuge tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the volume of Wash Buffer W7 specified in the table above. Important: Do not let the beads dry out.

  3. Remove the tube from the magnetic separator and resuspend the beads in the wash buffer by pipetting gently up and down.

  4. When the incubation time of the lysate has ∼4 minutes left (Step 5), repeat the wash procedure in Steps 2 and 3 above one more time, and then proceed to Step 5.

  5. Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette and discard the solution, and immediately add the lysate from Cell Lysis, Step 5, above, and the heated Binding Buffer B6 from Preparing the Binding Buffer and Lysis Buffer, Step 1.

  6. Remove the tube from the magnetic separator and resuspend the beads completely in the solution by pipetting up and down.

  7. Cap the tube and place in the heat block or thermal cycler. Incubate at 65–70°C for 2–5 minutes.

  8. Transfer the tube to a rotator and rotate the sample for 10 minutes at room temperature. Proceed to Wash and Elution Procedure below.

Wash and Elution Procedure

  1. Insert the tube from Prewash and Binding Procedure, Step 8, above, in the magnetic separator. Visually inspect the beads until they are clearly separated (∼2–5 minutes).   Note: Beads may take longer to separate depending on the viscosity of the lysate and the abundance of non-sheared genomic DNA.

  2. When the beads are separated, remove and save the supernatant.   Note: For rare samples, if your yield is low, you may want to preserve this supernatant and attempt to perform a separate isolation with it.

  3. To the beads, immediately add the volume of Wash Buffer W6 specified in the table on above.

  4. Remove the tube from the magnetic separator and resuspend the beads by pipetting gently up and down.

  5. If you are using > 1 × 106 cells and/or performing downstream RT-PCR: Add 1 μl (1 unit) of DNase I, Amplification Grade, per 100 μl of Wash Buffer W6 to the tube. Mix by pipetting gently up and down and incubate at 25°C for 5–10 minutes.

  6. Reinsert the tube in the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Carefully remove the wash buffer completely.

  7. To the beads, immediately add the volume of Wash Buffer W7 specified in the table above.

  8. Remove the tube from the magnetic separator and resuspend the beads by pipetting gently up and down.

  9. Reinsert the tube in the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Be careful to completely remove the wash buffer.

  10. Repeat Steps 7–9 using Wash Buffer W7 two more times, then proceed to Step 11.

  11. If you started with < 1 × 106 cells, immediately add 5-20 μl of RNase-free water. If you started with > 1 × 106 cells, immediately add 20–50 μl of RNase-free water. Remove the tube from the magnetic separator and thoroughly resuspend the beads by pipetting gently up and down. Incubate at 37°C for 2–5 minutes. Use the longer incubation time for larger samples.

  12. Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5-2 minutes), remove and save the supernatant.

  13. Important: The supernatant contains the isolated mRNA. Do not discard.

  14. Repeat Steps 11–13, using 5 μl of RNase-free water for < 1 × 106 cells and 10 μl of RNase-free water for > 1 × 106 cells. Again, save the supernatant.

  15. Combine the supernatants from Steps 13 and 14. This is your isolated mRNA. Store mRNA at -80°C.

The mRNA quality and yield may be determined and spectrophotometry and agarose gel electrophoresis
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Isolating mRNA from Tissues

Introduction

This section provides procedures for isolating mRNA from tissue samples.

User Supplied Materials

  • Magnetic particle  separator (the Magna-Sep™ 6-hole MPS is available from Invitrogen
  • Heat block or thermal cycler, preheated to 65–70°C
  • Water bath or incubator at 45°C
  • RNase-free tubes
  • Homogenizer, motor-driven, compatible with microcentrifuge tube used to prepare tissue sample (various manufacturers)
  • Pipettes
  • Rotator
  • Tabletop microcentrifuge
  • Optional for Micro kit: DNase I, Amplification Grade (Catalog no. 18068-015)

Amounts of Beads and Buffers

The following table lists the amounts of FastTrack® MAG Beads, Lysis Buffer, Proteinase K, Binding Buffer B6, Wash Buffer W6, and Wash Buffer W7 to use with the specified amount of tissue.
Note:   The isolation procedure has five separate washes using Wash Buffer W7, so each volume is shown × 5.


Amount Micro   Maxi  
Tissue< 10 mg
10–50 mg
50–200 mg<200–500 mg*
FastTrack® MAG Beads20 μl20-50 μl50-100 μl100-200 μl
Lysis Buffer L4
100 μl
200 μl
500 μl
500 μl
Proteinase K
2.5 μl
5 μl
10 μl
10 μl
Binding Buffer B6100 μl200 μl500 μl500 μl
Wash Buffer W6
100 μl200 μl500 μl500 μl
Wash Buffer W7100 μl × 5200 μl × 5500 μl × 5500 μl × 5

*For tissue amounts > 200 mg, you can use more Lysis Buffer L4/Proteinase K and Binding Buffer B6 in multiple tubes or proportionally scale up to a larger tube.

  • The abundance of mRNA varies greatly in different tissues. Adjust the volumes accordingly.
  • To maximize the yield of mRNA from tissues, we suggest first isolating total RNA from the tissue using TRIzol® Reagent or another method and then isolating mRNA from the total RNA using this kit

Preparing Binding Buffer and Lysis Buffer

Preheat the Binding Buffer B6 and prepare the Lysis Buffer L4 as described below:

  1. Add the volume of Binding Buffer B6 specified in the table above to an RNase-free tube. Place the tube in a heat block or thermal cycler at 65–70°C.

  2. In a separate RNase-free tube, add the volume of Proteinase K specified in the table above to the specified volume of Lysis Buffer L4. Proceed with Preparing the Tissue Sample.

Important: 
Homogenize tissue in the presence of lysis buffer to ensure immediate inactivation of any RNases that are released as the cells lyse. Complete homogenization is critical for complete cell lysis and inactivation of RNases.

Before use, clean and wash the homogenizer tip and then autoclave and bake for 3 hours or overnight at 210°C.

Pipetting Liquid from Tubes

When pipetting liquid from a tube in the magnetic separator, always point the pipette tip toward the opposite side of the tube bottom from the pellet to avoid touching the beads.


Preparing the Tissue Sample

Prepare the tissue sample as described below.

  1. Add the Lysis Buffer L4 plus Proteinase K from Step 2, above, to your frozen or fresh tissue sample in a sterile microcentrifuge tube. We recommend using a 1.5-ml tube for < 50 mg of tissue and a 12-ml 2059 tube for > 50 mg of tissue.

  2. Immediately homogenize the tissue using a motor-driven homogenizer that is compatible with your microcentrifuge tube. Start at a low speed and gradually increase speed until the homogenate appears smooth with no visible particulate matter (~15-30 seconds). Keep foaming to a minimum by adjusting the speed. Note: Thorough homogenization is required for maximum mRNA yield.

  3. Transfer the tube to a microcentrifuge and spin at maximum speed for 5 minutes at room temperature.

  4. Carefully transfer the supernatant containing your sample to a fresh microcentrifuge tube.

  5. Incubate the sample at 45°C in a water bath or incubator for 10 minutes. While the sample is incubating, proceed to Prewash and Binding Procedure below.

When following the procedures in this section, be careful to never let the beads dry out.

Prewash and Binding Procedure

While the sample is incubating (above), begin the procedure below to wash the beads and bind the sample:

  1. Thoroughly resuspend the FastTrack® MAG Beads by pipetting them gently up and down. Transfer the amount of resuspended beads specified in the table to an RNase-free microcentrifuge tube.

  2. Insert the microcentrifuge tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the volume of Wash Buffer W7 specified in the table. Important: Do not let the beads dry out.

  3. Remove the tube from the magnetic separator and resuspend the beads in the wash buffer by pipetting gently up and down.

  4. When the incubation time of the sample has ∼2 minutes left (Step 5, Preparing the Tissue Sample, previous page), repeat the wash procedure in Steps 2 and 3 above one time, and then proceed to Step 5 below.

  5. Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), pipette the liquid out of the tube and discard, and immediately add the sample from Step 5, previous page, and the heated Binding Buffer B6 from Step 1.

  6. Remove the tube from the magnetic separator and resuspend the beads completely in the solution by pipetting up and down.

  7. Cap the tube and place in the heat block or thermal cycler. Incubate at 65–70°C for 2–5 minutes.

  8. Transfer the tube to a rotator and rotate the sample for 10 minutes at room temperature. Proceed to Wash and Elution Procedure.

Wash and Elution Procedure

  1. Insert the tube from Washing and Binding Procedure, Step 8, previous page, in the magnetic separator. Visually inspect the beads until they are clearly separated (∼2–5 minutes).  Note:   Beads may take longer to separate depending on the viscosity of the sample and the abundance of non-sheared genomic DNA.

  2. When the beads are separated, remove and save the supernatant.  Note:   For rare samples, if your yield is low, you may want to preserve this supernatant and attempt to perform a separate isolation with it.

  3. To the beads, immediately add the volume of Wash Buffer W6 specified in the table

  4. Remove the tube from the magnetic separator and resuspend the beads in the wash buffer by pipetting gently up and down.

  5. If you are using > 50 mg tissue and/or performing downstream RT-PCR:   Add 1 μl (1 unit) of DNase I, Amplification Grade, per 100 μl of Wash Buffer W6 to the tube. Mix by pipetting gently up and down and incubate at 25°C for 5–10 minutes.

  6. Reinsert the tube in the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Be careful to completely remove the wash buffer.

  7. To the beads, immediately add the volume of Wash Buffer W7 specified in the table.

  8. Remove the tube from the magnetic separator and resuspend the beads by pipetting gently up and down.

  9. Reinsert the tube in the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove the wash buffer from the tube and discard. Be careful to completely remove the wash buffer.

  10. Repeat Steps 7–9 using Wash Buffer W7 two more times, then proceed immediately to Step 11.

  11. If you started with < 50 mg of tissue, immediately add 5–20 μl of RNase-free water. If you started with > 50 mg of tissue, immediately add 20–50 μl of RNase-free water.

  12. Remove the tube from the magnetic separator and thoroughly resuspend the beads by pipetting gently up and down. Incubate at 37°C for 2–5 minutes. Use the longer incubation time for larger samples.

  13. Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid (∼0.5–2 minutes), remove and save the supernatant.  Important: The supernatant contains the isolated mRNA. Do not discard.

  14. Repeat Steps 11–13, using 5 μl of RNase-free water for < 50 mg of tissue and 10 μl of RNase-free water for > 50 mg of tissue. Again, save the supernatant.

  15. Combine the supernatants from Steps 13 and 14. This is your isolated mRNA. Store mRNA at -80°C.

The mRNA quality and yield may be determined and spectrophotometry and agarose gel electrophoresis.
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Determining mRNA Yield and Quality

Determining of mRNA Yield

The following general protocol may be used to calculate the yield of mRNA using A260 absorbance:

  1. Aliquot 2 μl of the isolated mRNA into a clean UV cuvette and add 198 μl of TE Buffer for a 1:100 dilution.

  2. Blank a UV/visible spectrophotometer using TE Buffer, and then measure the sample at 260 nm.

  3. The A260 reading should fall within the standard specification for the spectrophotometer (typically 0.01–1.0 OD). If it falls outside this range, adjust the dilution and rescan. If the A260 reading is too low, use a lower dilution; if it’s too high, use a higher dilution.

  4. Calculate the yield of mRNA using the formula below:

mRNA yield (μg/μl) = A260 × 0.04 μg/μl RNA × Dilution factor
The dilution factor is 100 for the dilution described above. For example, if you diluted 2 μl of mRNA at 1:100, and the A260 is 0.5, then 0.5 × 0.04 μg/μl RNA × 100 = 2 μg/μl.

Analyzing mRNA Quality

The quality of the isolated mRNA may be determined by agarose gel electrophoresis. The mRNA appears as a smear from 500 bp to 8 kb, with the highest intensity between ∼1–3 kb. Any contaminating ribosomal RNA will appear as bands within the mRNA smear. These bands should be faint (< 20% higher intensity than the rest of the smear).

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Troubleshooting

The table below describes solutions to possible problems you may have with the kit. For additional assistance, contact Invitrogen Technical Support

ProblemSolution
Low yields of mRNA

  • Cells or tissues are not sufficiently homogenized
  • Cells or tissues are not completely lysed
  • mRNA is not completely eluted from the magnetic beads 
  • Homogenize cells/tissues until no or minimal cell clumps or tissue chucks are visible. We recommend use motor-driven homogenizers. Cells or tissues are not completely lysed
  • Reduce the amount of starting material, or increase the amount of lysis buffer. mRNA is not completely eluted from the magnetic beads
  • Wash the beads one more time using RNase-free water.
mRNA is degraded
  • Make sure that pipette tips, tubes, and other materials are RNase-free. Use RNase AWAY® Reagent to clean materials used for RNA isolation.
  • Add an RNase inhibitor (e.g., RNaseOUT™) to the starting material (e.g., the cell medium). Dead cells/tissues can release RNases that cause RNA degradation. Minimize dead cells/tissues and immediately freeze samples to prevent RNA degradation.
  • Check the quality of the purified total RNA on a gel. The 28S band should have a higher intensity than the 18S band, and the 18S band should have a higher intensity than the 5S band. If you see an obvious smear at low molecular weight (between 5S to 18S rRNA), it indicates that the total RNA is partially degraded.
Samples have a low abundance of RNAUse more starting material.
mRNA is contaminated with rRNA

Poor removal of rRNA during wash steps
Wash the beads 1 or 2 more times.


Perform the full procedure again, using the isolated mRNA from the first procedure.
mRNA is contaminated with genomic DNA

Poor removal of genomic DNA during the wash steps



Use the optional DNase I digestion step to remove genomic DNA contamination. Be sure to use Amplification Grade DNase I.
Viscous cell lysate

Sample is too large (mRNA will bind to oligo(dT) beads if the viscosity is reduced) Reduce the sample size, or split the sample.

Sample contains large amounts of DNA
Reduce the sample size, or split the sample.







Split the sample as above and shear the DNA thoroughly using a 21-guage needle on a 1-ml syringe.
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References

  1. Morrissey, D. V., Lombardo, M., Eldredge, J. K., Kearney, K. R., Groody, E. P., and Collins, M. L. (1989) Nucleic Acid Hybridization Assays Employing dA-tail Capture Probes. Anal. Biochem. 181, 345-359

  2. Stone, B. B., Cohen, S. P., Breton, G. L., Nietupski, R. M., Pelletier, D. A., Fiandaca, M. J., Moe, J. G., Smith, J. H., Shah, J. S., and Weisburg, W. G. (1996) Detection of rRNA from Four Respiratory Pathogens Using an Automated Qβ Replicase Assay. Molecular and Cellular Probes 10, 359-370
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25-0754   Version C   17-Apr-2007