Mag-Bind®XP FFPE RNA Kit Mag-Bind®XP FFPE - Omega Bio-Tek

Mag-Bind® XP FFPE RNA Kit

M2595-00 5 prepsM2595-01 50 prepsM2595-02 200 preps

Mag-Bind® XP FFPE RNA 96 Kit

M2596-00 1 x 96 prepsM2596-01 4 x 96 prepsM2596-02 20 x 96 preps

September 2012

1

Mag-Bind® XP FFPE RNA Kit Mag-Bind® XP FFPE RNA 96 Kit

Table of Contents

Introduction and Principle........................................................2Starting Materials..........................................................................2Kit Contents.....................................................................................3Preparing Reagents / Storage .................................................4-5 Mag-Bind® XP FFPE RNA Protocol.........................................6 Mag-Bind® XP FFPE RNA Protocol with Xylene............10 Mag-Bind® XP FFPE RNA 96 Protocol.................................14Mag-Bind® XP FFPE RNA 96 Protocol with Xylene.......18Troubleshooting Guide.............................................................23

Manual Revision: September 2012

Innovations in nucleic acid isolation

2

Introduction

The Mag-Bind® XP FFPE RNA kit and the Mag-Bind® XP FFPE RNA 96 kit provide a rapid and easy method for the isolation of total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. Due to fixation and embedding procedures, nucleic acids in FFPE samples are heavily fragmented and modified by formaldehyde. While the Mag-Bind® XP FFPE RNA kit and the Mag-Bind® XP FFPE RNA 96 kit are optimized to minimize the effect of the formaldehyde modification, it is not recommended to use the RNA purified with this kit for downstream applications that requires full length RNA.

Principle

The Mag-Bind® XP FFPE RNA kit and the Mag-Bind® XP FFPE RNA 96 kit combine high efficient binding properties of Mag-Bind® technology with a specially designed buffer system to isolate total RNA from FFPE tissue samples. There are two protocols included in this manual. The standard protocol uses a heating step instead of xylene to remove paraffin from the sample. The alternative protocol uses traditional xylene extraction to remove paraffin.

Samples are lysed in RML Buffer and digested with Proteinase K Solution. The lysate is mixed with XPL Enhancer and incubated in DNase I to remove DNA. After adjusting the binding conditions, RNA is bound to the surface of the Mag-Bind® Particles CNR. After two wash steps, purified RNA is eluted with DEPC Water.

Starting Materials Since standard formalin fixation and paraffin embedding procedures cause significant fragmentation of nucleic acids. We recommend following guidelines to limit the extent of DNA/RNA fragmentation: 1) Use 4-10% formalin to fixate tissue samples; 2) Limit the fixation time to 14-24 hours; 3) Completely dehydrate samples before embedding. Always use freshly cut sections of FFPE tissue for RNA isolation. For the first time user, we recommend to use less than 3-5 sections with thickness of 10 μm. Depending on the yield and purity obtained, it may be possible to increase the starting material.

New in this Edition:

• Proteinase K is now supplied in a liquid form eliminating the resuspension step prior to use. Proteinase K Solution can be stored at room temperature for 12 months.

• Proteinase Storage Buffer is no longer included in the kit.

3

Kit Contents

Mag-Bind® XP FFPE RNA KitProduct M2595-00 M2595-01 M2595-02

Preparations 5 50 200

Mag-Bind® Particles CNR 30 µL 270 µL 1.1 mL

RML Buffer 2 mL 20 mL 60 mL

XPL Enhancer Buffer 150 µL 1.5 mL 5 mL

XPN Buffer 2 mL 20 mL 80 mL

RNA Wash Buffer II 5 mL 12 mL 50 mL

LPA Buffer 55 µL 550 µL 2.2 mL

VHB Buffer 3 mL 9 mL 36 mL

DNase I 12 µL 80 µL 320 µL

Proteinase K Solution 110 µL 1.1 mL 4.4 mL

DEPC Water 1 mL 5 mL 15 mL

User Manual P P P

Mag-Bind® XP FFPE RNA 96 KitProduct M2596-00 M2596-01 M2596-02

Preparations 1 x 96 4 x 96 20 x 96

Mag-Bind® Particles CNR 0.6 mL 2.2 mL 11 mL

RML Buffer 35 mL 140 mL 700 mL

XPL Enhancer Buffer 3 mL 12 mL 50 mL

XPN Buffer 25 mL 100 mL 2 x 250 mL

RNA Wash Buffer II 25 mL 100 mL 2 x 200 mL

LPA Buffer 1.1 mL 4.4 mL 22 mL

VHB Buffer 18 mL 75 mL 300 mL

DNase I 160 µL 4 x 160 µL 20 x 160 µL

Proteinase K Solution 2.2 mL 9 mL 45 mL

DEPC Water 20 mL 40 mL 160 mL

User Manual P P P

4

Please take a few minutes to read this manual thoroughly to become familiar with the protocol before beginning the procedure. Prepare all materials required before starting to minimize RNA degradation. Wear gloves/protective goggles and take great care when working with chemicals

• Dilute RNA Wash Buffer II with 100% ethanol as follows and store at room

temperature.

Kit 100% Ethanol to be Added

M2595-00 20 mL

M2595-01 48 mL

M2595-02 200 mL


M2596-00 100 mL

M2596-01 400 mL

M2596-02 800 mL per bottle

• Dilute XPN Buffer with isopropanol as follows and store at room temperature.

Kit Isopropanol to be Added

M2595-00 4 mL

M2595-01 40 mL

M2595-02 160 mL

Kit Isopropanol to be Added

M2596-00 50 mL

M2596-01 200 mL

M2596-02 500 mL per bottle

Preparing Reagents

5

Preparing Reagents/Storage

Storage and Stability

All of the Mag-Bind® XP FFPE RNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows. Proteinase K Solution can be stored at room temperature for up to 12 months. For long-term storage, store Proteinase K Solution at 2-8°C. The Mag-Bind® Particles CNR should be stored at 2-8°C for long-term storage. DNase I should be stored at -20°C. During shipping and storage, crystals may form in the RML Buffer, simply warm to 37°C to dissolve.

• Dilute VHB Buffer with 100% ethanol and store at room temperature.


M2595-00 7 mL

M2595-01 21 mL

M2595-02 84 mL


M2596-00 42 mL

M2596-01 175 mL

M2596-02 700 mL

6

Mag-Bind® XP FFPE RNA - 1.5 mL tube (M2595)

Materials and Equipment to be Provided by User:

• Centrifuge capable of ≥13,000 x g• Magnetic separation device for 1.5 mL tubes (Cat# MSD-02)• Water bath or heat block capable of 55°C• Water bath or heat block capable of 80°C• Vortexer• Nuclease-free 1.5 mL microcentrifuge tubes• RNase-free filter pipette tips• 100% Ethanol• Isopropanol

Before Starting: • Prepare Buffers according to Preparing Reagents section on Pages 4-5• Set water bath or heat block to 55°C• Set water bath or heat block to 80°C

1. Add 250 μL RML Buffer into a 1.5 mL microcentrifuge tube.

2. Cut 3-8 paraffin sample sections between 5-10 μm.

Note: Do not use the first 2-3 sections.

3. Immediately add 2-5 sections to the RML Buffer. Vortex or shake for 30 seconds to mix thoroughly.

4. Centrifuge at maximum speed (≥13,000 x g) for 20 seconds.

5. Incubate at 80°C for 15 minutes. Mix the sample a few times by gently shaking the tube 2-3 times. Make sure that the tissue sections stay submerged in the solution.

6. Cool the sample to room temperature.

Mag-Bind® XP FFPE RNA Protocol

7

7. Add 20 µL Proteinase K Solution.

8. Incubate at 55°C for 15-30 minutes with occasional mixing. If necessary, extend the incubation to 1-3 hours or until the tissue is completely lysed.

9. Incubate at 80°C for 15 minutes.


11. Add 20 µL XPL Enhacer Buffer and 1.5 µL DNase I. Pipet up and down 5 times to mix thoroughly.

12. Let sit at room temperature for 15 minutes.

13. Centrifuge at maximum speed (≥13,000 x g) for 5 minutes. The paraffin will form a thin layer on top of the lysate solution.

14. Transfer 200 μL cleared lysate into a new 1.5 mL microcentrifuge tube.

Tip: Use a 1 mL pipette tip or large orifice tip to penetrate the paraffin layer.

15. Add 5 µL Mag-Bind® Particles CNR and 600 µL XPN Buffer. Vortex for 30 seconds or pipet up and down 10-20 times to mix thoroughly.

Note: If the RNA content from sample is expected low or miRNA is the target, add 10 µL LPA Buffer.

Note: XPN Buffer must be diluted with isopropanol prior to use. Please see Page 4 for instructions.

16. Let sit at room temperature for 5-10 minutes.

17. Place the tube on a magnetic separation device to magnetize the Mag-Bind® Particles CNR. Let sit at room temperature until the Mag-Bind® Particles CNR are completely cleared from solution.


8

18. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind® Particles CNR.

19. Remove the tube containing the Mag-Bind® Particles CNR from the magnetic separation device.

20. Add 400 µL VHB Buffer. Resuspend the Mag-Bind® Particles CNR by vortexing for 20

seconds or pipetting up and down 20 times.

Note: VHB Buffer must be diluted with ethanol prior to use. Please see Page 5 for instructions.




24. Add 400 µL RNA Wash Buffer II. Resuspend the Mag-Bind® Particles CNR by vortexing for 20 seconds or pipetting up and down 20 times.



27. Repeat Steps 23-26 for a second RNA Wash Buffer II wash step.

28. Leave the tube on the magnetic separation device for 5-10 minutes to air dry the Mag-Bind® Particles CNR. Remove any residual liquid with a pipettor.


9

29. Add 30-50 µL DEPC Water. Resuspend the Mag-Bind® Particles CNR by vortexing for 20 seconds or pipetting up and down 20 times.



32. Transfer the cleared supernatant containing purified RNA to a clean 1.5 mL microcentrifuge tube.

33. Store the RNA at -80°C.


10

Mag-Bind® XP FFPE RNA with Xylene - 1.5 mL tube (M2595) Note: The following protocol uses xylene to remove paraffin from the FFPE sample. Use a fume hood and take proper precautions during the xylene extraction.


• Centrifuge capable of ≥13,000 x g• Magnetic separation device for 1.5 mL tubes (Cat# MSD-02)• Water bath or heat block capable of 55°C• Water bath or heat block capable of 80°C• Vortexer• Nuclease-free 1.5 mL microcentrifuge tubes• RNase-free filter pipette tips• Xylene• 100% Ethanol• Isopropanol


1. Add 1 mL xylene into a 1.5 mL microcentrifuge tube.

2. Cut 3-8 paraffin sample sections between 5-10 μm. Note: Do not use the first 2-3 sections.

3. Immediately add 2-5 sections to the xylene.

4. Vortex for 20 seconds to mix thoroughly.


Mag-Bind® XP FFPE RNA Protocol with Xylene

11

6. Centrifuge at maximum speed (≥13,000 x g) for 5 minutes to pellet the tissue. Note: If the tissue does not form a tight pellet, centrifuge for an additional 3 minutes.

7. Aspirate and discard the xylene. Do not disturb the tissue pellet.

8. Add 1 mL 100% ethanol. Vortex for 20 seconds to mix thoroughly.

9. Centrifuge at maximum speed (≥13,000 x g) for 5 minutes to pellet the tissue. The pellet should appear opaque.

10. Aspirate and discard the ethanol. Do not disturb the tissue pellet. Remove any liquid drops with a pipette.

11. Repeat Steps 8-10 for a second ethanol wash step.

12. Air dry the tissue pellet for 10-20 minutes. Note: It is critical to completely dry the sample before the next Proteinase K digestion step. Residual ethanol will affect the efficiency of the Proteinase K digestion. If a vacuum oven is available, place the tube in the vacuum oven preset at 45°C for 10-20 minutes.

13. Add 250 μL RML Buffer and 20 μL Proteinase K Solution. Resuspend the pellet by vortexing for 20 seconds or pipetting up and down 20 times.






12


19. Centrifuge at maximum speed (≥13,000 x g) for 5 minutes.

20. Transfer 200 μL cleared lysate into a new 1.5 mL microcentrifuge tube.

21. Add 5 µL Mag-Bind® Particles CNR and 600 µL XPN Buffer. Vortex for 30 seconds or pipette up and down 10-20 times to mix thoroughly.







26. Add 400 µL VHB Buffer. Resuspend the Mag-Bind® Particles CNR by vortexing for 20 seconds or pipetting up and down 20 times.




13







34. Leave the tube on the magnetic separation device for 5-10 minutes to air dry the Mag-Bind® Particles CNR. Remove any residual liquid with a pipettor.




38. Transfer the cleared supernatant containing purified RNA to a clean 1.5 mL microcentrifuge tube.



14

Mag-Bind® XP FFPE RNA 96 Protocol

Mag-Bind® XP FFPE RNA 96 - 96-well plate (M2596) Materials and Equipment to be Provided by User:

• Centrifuge with swing-bucket rotor capable of ≥4,000 x g• Magnetic separation device for 96-well plates (Cat# MSD-01B or MSD-01)• Water bath or heat block capable of 55°C• Water bath or heat block capable of 80°C• Vortexer• Nuclease-free 1.2 mL or 2 mL 96-well round-well plates• Nuclease-free 96-well microplates• RNase-free filter pipette tips• 100% Ethanol• Isopropanol• Sealing film


1. Add 250 μL RML Buffer into each well of a 1.2 mL or 2 mL 96-well round-well plate.

2. Cut 3-8 paraffin sample sections between 5-10 μm.

Note: Do not use the first 2-3 sections from the sample block.

3. Immediately add 2-5 sections to the RML Buffer.

4. Centrifuge at 4,000 x g for 2 minutes at room temperature.

5. Incubate at 80°C for 15 minutes. Mix the sample a few times by gently shaking the plate 2-3 times. Make sure that the tissue sections stay submerged in the solution.


15

7. Add 20 µL Proteinase K Solution.



10. Cool the samples to room temperature.



13. Centrifuge at 4,000 x g for 5 minutes. The paraffin will form a thin layer on top of the lysate solution.

14. Transfer 200 μL cleared lysate into a new 96-well round-well plate.

Tip: Use a 1 mL pipette tip or large orifice tip to penetrate the paraffin layer.






16

17. Place the plate on a magnetic separation device to magnetize the Mag-Bind® Particles CNR. Let sit at room temperature until the Mag-Bind® Particles CNR are completely cleared from solution.

Note: If using the MSD-01 magnetic separation device, a 500 µL 96-well plate (EZ960-01/02) is required for the rest of the protocol. Since the total volume of the sample is around 850 µL, this particular magnetic separation device requires the sample be transferred twice to process the whole sample.


19. Remove the plate containing the Mag-Bind® Particles CNR from the magnetic separation device.









17



28. Leave the plate on the magnetic separation device for 5-10 minutes to air dry the Mag-Bind® Particles CNR. Remove any residual liquid with a pipettor.




32. Transfer the cleared supernatant containing purified RNA to a nuclease-free 96-well microplate. Seal with sealing film.



18

Mag-Bind® XP FFPE RNA 96 Protocol with Xylene

Mag-Bind® FFPE RNA 96 with Xylene- 96-well plate (M2596) Note: The following protocol uses xylene to remove paraffin from the FFPE sample. Use a fume hood and take proper precautions during the xylene extraction.


• Centrifuge with swing-bucket rotor capable of 4,000 x g• Magnetic separation device for 96-well plates (Cat# MSD-01B or MSD-01)• Water bath or heat block capable of 55°C• Water bath or heat block capable of 80°C• Vortexer• Nuclease-free 1.2 mL or 2 mL 96-well round-well plates• Nuclease-free 96-well microplates• RNase-free filter pipette tips• 100% Ethanol• Isopropanol• Xylene• Sealing film


1. Add 1 mL xylene into each well of a 1.2 mL or 2 mL 96-well round-well plate.

2. Cut 3-8 paraffin sample sections between 5-10 μm. Note: Do not use the first 2-3 sections.

3. Immediately add 2-5 sections to the xylene.

4. Vortex for 20 seconds to mix thoroughly.


19

6. Centrifuge at 4,000 x g for 5 minutes to pellet the tissue. Note: If the tissue does not form a tight pellet, centrifuge for an additional 3 minutes.

7. Aspirate and discard the xylene. Do not disturb the tissue pellet.

8. Add 1 mL 100% ethanol. Vortex for 20 seconds to mix thoroughly.

9. Centrifuge at 4,000 x g for 5 minutes to pellet the tissue. The pellet should appear opaque.

10. Aspirate and discard the ethanol. Do not disturb the tissue pellet. Remove any liquid drops with a pipette.

11. Repeat Steps 8-10 for a second ethanol wash step.

12. Air dry the tissue pellet for 10-20 minutes. Note: It is critical to completely dry the sample before the next Proteinase K digestion step. Residual ethanol will affect the efficiency of the Proteinase K digestion. If a vacuum oven is available, place the tube in the vacuum oven preset at 45°C for 10-20 minutes.

13. Add 250 μL RML Buffer and 20 μL Proteinase K Solution. Resuspend the pellet by vortexing for 20 seconds or pipetting up and down 20 times.




17. Add 20 µL XPL Enhacer Buffer and 1.5 µL DNase I. Pipette up and down 5 times to mix thoroughly.


20


19. Centrifuge at 4,000 x g for 5 minutes.

20. Transfer 200 μL cleared lysate into a new 96-well round-well plate.






Note: If using the MSD-01 magnetic separation device, a 500 µL 96-well plate (EZ960-01/02) is required for the rest of the protocol. Since the total volume of the sample is around 850 µL, this particular magnetic separation device requires the sample be transferred twice to process the whole sample.




21










34. Leave the plate on the magnetic separation device for 5-10 minutes to air dry the Mag-Bind® Particles CNR. Remove any residual liquid with a pipettor.


22




38. Transfer the cleared supernatant containing purified RNA to a nuclease-free 96-well microplate. Seal with sealing film.



23

Troubleshooting Guide

Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact our technical support staff, toll free, at 1-800-832-8896.

Possible Problems and Suggestions

Problem Likely Cause Suggestions

Low RNA yields

Incomplete re-suspension of magnetic particles

Resuspend the magnetic particles by vortexing before use.

RNA degraded during sample storage

Do not over-fix the samples in formalin. Make sure the samples are processed immediately after sectioning.

VHB Buffer and RNA Wash Buffer II were not prepared correctly

Prepare VHB Buffer and RNA Wash Buffer II according to the instructions on Pages 4-5.

Loss of magnetic beads during operation Increase the beads collection time.

XPN Buffer was not diluted with ethanol

Prepare XPN Buffer according to the instructions on Page 4.

Problem with downstream application

Degraded RNA Do not exceed 15 minutes for the 80°C incubation step.

Carryover of the magnetic particles in the elution

Carryover of magnetic particles in the eluted RNA will not effect downstream applications

To remove the carryover magnetic particles from the eluted RNA, simply magnetize the magnetic particles and carefully transfer the RNA eluate to a new tube or plate.

24

Notes:

Mag-Bind®XP FFPE RNA Kit Mag-Bind®XP FFPE - Omega Bio-Tek

Documents