FastTrack MAG mrna Isolation Kits

Transcription

1 USER GUIDE FastTrack MAG mrna Isolation Kits For isolating high-quality mrna from total RNA, cells, and tissue Catalog Numbers K and K Document Part Number Publication Number MAN Revision 2.0 For Research Use Only. Not for use in diagnostic procedures.

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3 Contents Experienced Users Procedure to Isolate mrna from Total RNA... 4 Experienced Users Procedure to Isolate mrna from Cells... 6 Experienced Users Procedure to Isolate mrna from Tissues... 8 Kit Contents and Storage Introduction System Overview Handling RNA Methods Isolating mrna from Total RNA Isolating mrna from Cells Isolating mrna from Tissues Determining mrna Yield and Quality Troubleshooting Appendix Accessory Products Technical Support References

4 Experienced Users Procedure to Isolate mrna from Total RNA Introduction Step Total RNA Protocol 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. Procedure 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 μg 1 50 μg μg μg FastTrack MAG Beads 20 μl 50 μl 100 μl 200 μl Binding Buffer B6 100 μl 200 μl 500 μl 500 μl Wash Buffer W7 100 μl μl μl μl 6 1. Isolate total RNA as described on page 15. Perform DNase I digestion, if you wish to use the mrna for RT-PCR. 2. Preheat the specified volume of Binding Buffer B6 (see table this page) at 65 C to 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 table) to an RNase-free microcentrifuge tube. 5. Insert the tube into the magnetic particle separator (MPS) for 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 minutes. Remove and discard the supernatant. 4

5 Experienced Users Procedure to Isolate mrna from Total RNA, Continued Step Total RNA Protocol, continued Procedure 10. Immediately add the total RNA sample from Step 3, and the heated Binding Buffer B6 from Step 2, previous page. 11. Remove the tube from the MPS and resuspend beads by pipetting gently up and down. 12. Incubate the capped tube at 65 C to 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 minutes. Remove and save the supernatant. 15. Immediately add the specified volume of Wash Buffer W Remove the tube from the MPS and resuspend the beads by pipetting gently up and down. 17. Reinsert the tube in the MPS for minutes. Remove and discard the wash buffer from the tube. 18. Repeat Steps three more times. 19. Immediately add 5 20 μl RNase-free water for <50 μg total RNA starting amount or add 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 minutes. Remove and save the supernatant. Important: The supernatant contains the isolated mrna. 22. Repeat Steps 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. 5

6 Experienced Users Procedure to Isolate mrna from Cells Introduction Step Cells Protocol 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. Procedure Use the listed amount of reagents with the specified amount of cells for the purification procedure described below. Amount Micro Maxi Cells < FastTrack MAG Beads 20 μl μl μl μl Binding Buffer B6 100 μl 200 μl 500 μl 500 μl Wash Buffer W6 100 μl 200 μl 500 μl 500 μl Wash Buffer W7 100 μl μl μl μl 5 1. Harvest cells and prepare cell lysate as described on page Preheat the specified volume of Binding Buffer B6 (see table) at 65 C to 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 table) to an RNase-free microcentrifuge tube. 4. Insert the tube into the MPS for 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 minutes. Remove and discard the supernatant. 9. Immediately add the cell lysate (page 20), and the heated Binding Buffer B6 from Step 2. 6

7 Experienced Users Procedure to Isolate mrna from Cells, Continued Step Cell Protocol, continued Procedure 10. Remove the tube from MPS and resuspend the beads by pipetting up and down. 11. Incubate the capped tube at 65 C to 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 W Remove the tube from the MPS and resuspend the beads by pipetting. 16. For > 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 minutes. Remove and discard the wash buffer from the tube. 18. Immediately add the specified volume of Wash Buffer W Remove the tube from the MPS and resuspend the beads by pipetting gently up and down. 20. Insert the tube in the MPS for minutes. Remove and discard the wash buffer. 21. Repeat Steps using Wash Buffer W7 two more times. 22. Immediately add 5 20 μl RNase-free water for a starting amount of < cells or add μl RNase-free water for a starting amount of > 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 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 < cells and 10 μl RNase-free water for > cells. 26. Combine the supernatants from Steps 24 and 25 containing your isolated mrna. Store mrna at 80 C. 7

8 Experienced Users Procedure to Isolate mrna from Tissues Introduction Step Tissue Protocol 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. Procedure Use the listed amount of reagents with the specified amount of tissue for the purification procedure described below. Amount Micro Maxi Tissue <10 mg mg mg mg FastTrack MAG Beads 20 μl μl μl μl Binding Buffer B6 100 μl 200 μl 500 μl 500 μl Wash Buffer W6 100 μl 200 μl 500 μl 500 μl Wash Buffer W7 100 μl μl μl μl 5 1. Harvest tissue and prepare tissue lysate as described on page Preheat the specified volume of Binding Buffer B6 (see table) at 65 C to 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 table) to an RNase-free microcentrifuge tube. 4. Insert the tube into the MPS for 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 minutes. Remove and discard the supernatant. 9. Immediately add the tissue lysate (page 26), and the heated Binding Buffer B6 from Step 2. 8

9 Experienced Users Procedure to Isolate mrna from Tissues, Continued Step Tissue Protocol, continued Procedure 10. Remove the tube from MPS and resuspend the beads by pipetting up and down. 11. Incubate the capped tube at 65 C to 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 W 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 minutes. Remove and discard the wash buffer from the tube. 18. Immediately add the specified volume of Wash Buffer W Remove the tube from the MPS and resuspend the beads by pipetting gently up and down. 20. Insert the tube in the MPS for minutes. Remove and discard the wash buffer from the tube. 21. Repeat Steps using Wash Buffer W7 two more times. 22. Immediately add 5 20 μl RNase-free water for <50 mg tissue starting material or add 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 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. 9

10 Kit Contents and Storage Shipping and Storage All components of the FastTrack MAG Micro mrna Isolation Kit (Cat. no. K ) and the FastTrack MAG Maxi (Cat. no. K ) mrna Isolation Kit are shipped at room temperature. Upon receipt, store all components at room temperature. Note: Do not freeze the beads. Kit Configurations The FastTrack MAG mrna Isolation Kits are designed to isolate mrna from different size sample ranges. The following table shows the maximum sample size for each kit (based on the amount of beads provided and the number of reactions specified). Maximum Sample Size Total RNA Cells Tissue Micro Kit 50 μg mg Maxi Kit 2000 μg mg Micro Kit Components The FastTrack MAG Micro mrna Isolation Kit provides reagents for 12 reactions using 50 μl of beads per reaction. Component FastTrack MAG beads Lysis Buffer L4 Proteinase K, 20 mg/ml in storage buffer (proprietary) Binding Buffer B6 Wash Buffer W6 Wash Buffer W7 RNase-Free Water Amount 600 μl 3 ml 75 μl 3 ml 3 ml 18 ml 750 μl 10

11 Kit Contents and Storage, Continued Maxi Kit Components The FastTrack MAG Maxi mrna Isolation Kit provides reagents for 6 reactions using 200 μl of beads per reaction. Component FastTrack MAG beads Lysis Buffer L4 Proteinase K, 20 mg/ml in storage buffer (proprietary) Binding Buffer B6 Wash Buffer W6 Wash Buffer W7 RNase-Free Water DNase I, Amplification Grade (1 U/μL) Amount 1.2 ml 4.8 ml 96 μl 4.8 ml 4.8 ml 28.8 ml 750 μl 100 μl 11

12 System Overview Introduction Introduction 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 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 (< 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 minutes Prewash FastTrack MAG Beads and bind sample minutes Wash FastTrack MAG Beads minutes Elute sample 5 10 minutes 12

13 System Overview, Continued 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%) cells 1 μg 1 g tissue 5 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 Life Technologies that can hold mL tubes (Cat. no. K ). You may also use separators from other manufacturers. 13

14 Handling RNA General Handling of RNA When working with RNA: Do not use plasticware or glassware without first eliminating possible ribonuclease contamination. 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. See page 33 for ordering information. 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. 14

15 Methods Isolating mrna from Total RNA Introduction User Supplied Materials Isolating Total RNA Note Amounts of Beads and Buffers This section provides procedures for isolating mrna from total RNA. Total RNA purification reagent/system Magnetic particle separator (the Magna-Sep 6-hole MPS is available from Life Technologies; see ordering information on page 33) Heat block or thermal cycler preheated to 65 C to 70 C RNase-free 1.5-mL tubes Pipettes Rotator Tabletop microcentrifuge To isolate total RNA, we recommend TRIzol Reagent (Cat. no and ), the PureLink RNA Mini Kit (Cat. no A), or the PureLink Pro 96 total RNA Purification Kit (Cat. no A). 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. 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 μg 1 50 μg μg μg FastTrack MAG Beads 20 μl 50 μl 100 μl 200 μl* Binding Buffer B6 100 μl 200 μl 500 μl 500 μl Wash Buffer W7 100 μl μl μl 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). 15

16 Isolating mrna from Total RNA, Continued Before Proceeding Before proceeding with the protocol, preheat the Binding Buffer B6 and add water to the total RNA sample as described. While the buffer is heating, proceed with the Prewash and Binding Procedure, page Add the volume of Binding Buffer B6 specified in the table on page 15 to a 1.5-mL RNase-free tube. Place the tube in a heat block or thermal cycler at 65 C to 70 C. 2. 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. Bead pellet 16

17 Isolating mrna from Total RNA, Continued Prewash and Binding Procedure While the Binding Buffer B6 is preheating (step 1 page 16), proceed with steps 1 4: 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 15 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 ( 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 15. 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 Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid ( minutes), pipette the liquid out of the tube and discard, and immediately add the total RNA sample from Step 2, page 16, and the heated Binding Buffer B6 from Step 1, page 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 C to 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, page

18 Isolating mrna from Total RNA, Continued Wash and Elution Procedure 1. Insert the tube from Prewash and Binding Procedure (Step 8, page 17) in the magnetic separator. When the beads are clearly separated ( 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 on page 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 ( 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 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 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 ( 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 on page

19 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 Life Technologies; see ordering information on page 33) Heat block or thermal cycler preheated to 65 C to 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 (Cat. no ) 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 < FastTrack MAG Beads 20 μl μl μl 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 B6 100 μl 200 μl 500 μl 500 μl Wash Buffer W6 100 μl 200 μl 500 μl 500 μl Wash Buffer W7 100 μl μl μl μl 5 *For cells amounts >5 10 7, you can use more Lysis Buffer L4/Proteinase K and Binding Buffer B6 in multiple tubes or proportionally scale up to a larger tube. 19

20 Isolating mrna from Cells, Continued Preparing Binding Buffer and Lysis Buffer Preheat the Binding Buffer B6 and prepare the Lysis Buffer L4 as described: 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 C to 70 C. 2. In a separate 1.5-mL RNase-free tube, add the volume of Proteinase K specified in the table (page 19) to the specified volume of Lysis Buffer L4. Proceed with Cell Lysis. Preparing Cells Collect, wash, and pellet cells according to your standard laboratory protocol. Cell Lysis Lyse cells as described. 1. To the cell pellet in a microcentrifuge tube, add the Lysis Buffer solution from Step 2, Preparing Binding Buffer and Lysis Buffer. 2. For < cells, pipette the lysate up and down at least 10 times. For > 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, page 21. 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. Bead pellet 20

21 Isolating mrna from Cells, Continued 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 (Step 5 page 20), proceed with the steps 1 4: 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 19 to an RNase-free microcentrifuge tube. 2. Insert the microcentrifuge tube into the magnetic separator. When the beads are clearly separated from the liquid ( 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 19. 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, page 20), repeat the wash procedure in Steps 2 and 3 one more time, and then proceed to Step Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid ( minutes), pipette and discard the solution, and immediately add the lysate from Cell Lysis, Step 5, page 20, and the heated Binding Buffer B6 from Preparing the Binding Buffer and Lysis Buffer, Step 1, page 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 C to 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, page

22 Isolating mrna from Cells, Continued Wash and Elution Procedure 1. Insert the tube from Prewash and Binding Procedure, Step 8, page 21, 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 nonsheared 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 page Remove the tube from the magnetic separator and resuspend the beads by pipetting gently up and down. 5. If you are using > 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 ( 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 on page 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 ( 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 If you started with < cells, immediately add 5 20 μl of RNase-free water. If you started with > cells, immediately add μl of RNase-free water. 22

23 Isolating mrna from Cells, Continued Wash and Elution Procedure, continued 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 ( 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 < cells and 10 μl of RNase-free water for > 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, as described on page

24 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 Life Technologies; see ordering information on page 33) Heat block or thermal cycler, preheated to 65 C to 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 (Cat no ) 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 mg mg mg* FastTrack MAG Beads 20 μl μl μl 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 B6 100 μl 200 μl 500 μl 500 μl Wash Buffer W6 100 μl 200 μl 500 μl 500 μl Wash Buffer W7 100 μl μl μl 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. 24

25 Isolating mrna from Tissues, Continued Note 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, as described starting on page 15. Preparing the Binding Buffer and Lysis Buffer Preheat the Binding Buffer B6 and prepare the Lysis Buffer L4 as described: 1. Add the volume of Binding Buffer B6 specified in the table on the previous page to an RNase-free tube. Place the tube in a heat block or thermal cycler at 65 C to 70 C. 2. In a separate RNase-free tube, add the volume of Proteinase K specified in the table on the previous page to the specified volume of Lysis Buffer L4. Proceed with Preparing the Tissue Sample, page 26. 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. Bead pellet 25

26 Isolating mrna from Tissues, Continued Preparing the Tissue Sample Prepare the tissue sample as described: 1. Add the Lysis Buffer L4 plus Proteinase K from Step 2, previous page, 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 motordriven 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, page

27 Isolating mrna from Tissues, Continued Important 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 (Step 5, page 26), begin the following procedure 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 on page 24 to an RNase-free microcentrifuge tube. 2. Insert the microcentrifuge tube into the magnetic separator. When the beads are clearly separated from the liquid ( 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 24. 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, page 26), repeat the wash procedure in Steps 2 and 3 one time, and then proceed to Step Insert the tube into the magnetic separator. When the beads are clearly separated from the liquid ( 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, page 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 C to 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, page

28 Isolating mrna from Tissues, Continued Wash and Elution Procedure 1. Insert the tube from Prewash and Binding Procedure, Step 8, page 27, 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 nonsheared 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 page 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 ( 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 on page 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 ( 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

29 Isolating mrna from Tissues, Continued Wash and Elution Procedure, continued 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 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 ( 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, as described on page

30 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 absorbance at 260 nm. 3. The A260 reading should fall within the standard specification for the spectrophotometer (typically 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) = A 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 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). In the 1.2% E-Gel agarose gel on the right, lane 1 and lane 2 each contain μg of mrna isolated from rat heart using the procedure described in this manual. Lane 3 was loaded with 2 μg of total RNA. 4k 3k 2k 1k 1Kb S 18S 5S 30

31 Troubleshooting Troubleshooting The following table describes solutions to possible problems you may have with the kit. For additional assistance, contact Life Technologies Technical Support (see page 34). Problem Cause Solution 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 mrna is degraded Samples have a low abundance of RNA Homogenize cells/tissues until no or minimal cell clumps or tissue chucks are visible. We recommend using motordriven homogenizers. Reduce the amount of starting material, or increase the amount of lysis buffer. Wash the beads one more time using RNase-free water. 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 Recombinant Ribonuclease Inhibitor) 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. Use more starting material. 31

32 Troubleshooting, Continued Problem Cause Solution Poor removal of Wash the beads 1 or 2 more times. rrna during wash steps mrna is contaminated with rrna Perform the full procedure again, using the isolated mrna from the first procedure. mrna is contaminated with genomic DNA Viscous cell lysate Poor removal of genomic DNA during the wash steps Sample is too large (mrna will bind to oligo(dt) beads if the viscosity is reduced) Sample contains large amounts of DNA Use the optional DNase I digestion step to remove genomic DNA contamination. Be sure to use Amplification Grade DNase I. 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. 32

33 Accessory Products Appendix Additional Products The following additional products are available from Life Technologies. To order, visit or contact Technical Support (see page 34). Product Quantity Catalog No. FastTrack MAG 96 mrna Isolation Kit 2 96 preps K Magna-Sep Magnetic Particle Separator Each K for use with 1.5-mL Tubes RNase AWAY Reagent 250 ml RNaseOUT Recombinant Ribonuclease 5000 units Inhibitor (40 units/μl) TRIzol Reagent 100 ml 200 ml DNase I, Amplification Grade 100 units PureLink RNA Mini Kit 50 preps A PureLink Pro 96 total RNA Purification Kit 4 plates A 33

34 Technical Support Obtaining Support For the latest services and support information for all locations, go to At the website, you can: Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions (FAQs) Submit a question directly to Technical Support (techsupport@lifetech.com) Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents Obtain information about customer training Download software updates and patches Safety Data Sheets (SDS) Safety Data Sheets (SDSs) are available at Certificate of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product. Certificates of Analysis are available on our website. Go to and search for the Certificate of Analysis by product lot number, which is printed on the box. Limited Product Warranty Life Technologies and/or its affiliate(s) warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on the Life Technologies web site at If you have any questions, please contact Life Technologies at 34

35 References 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, 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, Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. RNase AWAY is a registered trademark of Molecular Bio-Products, Inc. Trizol is a registered trademark of Molecular research Center, Inc. DISCLAIMER: LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. 35

36 Notes 36

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40 Headquarters 5791 Van Allen Way Carlsbad, CA USA Phone Toll Free in USA For support visit lifetechnologies.com/support or lifetechnologies.com 19 September 2012