Omega Mag-Bind FFPE RNA 96 User manual

Mag-Bind® FFPE RNA 96 Kit
M2551-00 1 x 96 preps
M2551-01 4 x 96 preps
March 2018

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Mag-Bind® FFPE RNA 96
Table of Contents
Introduction.....................................................................................2
Principle.............................................................................................2
Starting Materials...........................................................................2
Kit Contents......................................................................................3
Preparing Reagents/Storage..................................................4-5
Mag-Bind® FFPE RNA 96 Protocol............................................6
Mag-Bind® FFPE RNA 96 Protocol with Xylene.................10
Troubleshooting Guide..............................................................15
Manual Revision: March 2018

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Introduction
The Mag-Bind® FFPE RNA 96 kit provide a rapid and easy method for the isolation of
total RNA from formalin-xed, paran-embedded (FFPE) tissue sections. Due to xation
and embedding procedures, nucleic acids in FFPE samples are heavily fragmented and
modied by formaldehyde. While the Mag-Bind® FFPE RNA 96 kit are optimized to
minimize the eect of the formaldehyde modication, it is not recommended to use the
RNA puried with this kit for downstream applications that requires full length RNA.
Principle
The Mag-Bind® FFPE RNA 96 kit combine high ecient binding properties of Mag-Bind®
technology with a specially designed buer system to isolate total RNA sample from
FFPE sample. There are two protocols included in this manual. The standard protocol
uses a heating step instead of xylene to remove paran from the sample. The alternative
protocol uses traditional xylene extraction to remove paran.
Samples are rst lysed in RML Buer with digestion of Proteinase K. The lysate is then
mixed with MFB Buer and magnetic particles to bind the nucleic acid on the surface of
the Mag-Bind® Particles SC. Genomic DNA is removed by DNase I digestion. After two
wash steps, puried RNA is eluted with RNase-free water.
Starting Materials
Since standard formalin xation and paran embedding procedures cause signicant
fragmentation of nucleic acids. We recommend following guidelines to limit the extent
of DNA/RNA fragmentation: 1) Use 4-10% formalin to xate tissue samples; 2) Limit
the xation time to 14-24 hours; 3) Completely dehydrate samples before embedding.
Always use freshly cut sections of FFPE tissue for RNA isolation. For the rst 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:
March 2018
• The storage temperature for DNase I Digestion Buer has changed. DNase I Digestion
Buer should now be stored at -20°C along with the Mag-Bind® DNase I.
• The 200 prep size of M2551, Mag-Bind® FFPE RNA 96 Kit, has been discontinued and
is no longer available to purchase.
• M2555, Mag-Bind® FFPE RNA Kit, has been discontinued and is no longer available to
purchase.
January 2014
• DNase I has been renamed Mag-Bind® DNase I. This is a name change only.
• Proteinase K is now supplied in a liquid form eliminating the step to resuspend prior
to use. Proteinase K Solution can also be stored at room temperature for 12 months.
• Proteinase Storage Buer is no longer included in the kit.

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Kit Contents
Product Number M2551-00 M2551-01
Purications 1 x 96 4 x 96
Mag-Bind® Particles SC 2.2 mL 8.4 mL
RML Buer 35 mL 140 mL
MFB Buer 20 mL 80 mL
GFC Buer 10 mL 40 mL
RNA Wash Buer II 25 mL 2 x 50 mL
LPA Buer 1.1 mL 4.4 mL
DNase I Digestion Buer 2 x 5 mL 2 x 25 mL
Mag-Bind® DNase I 150 µL 4 x 150 µL
Proteinase K Solution (20 mg/mL) 3 mL 12 mL
DEPC Water 20 mL 40 mL
Instruction Booklet 1 1

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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
1. Dilute RNA Wash Buer II with 100% ethanol and store at room temperature.
Kit 100% Ethanol to be Added
M2551-00 100 mL
M2551-01 200 mL
2. Dilute GFC Buer with 100% ethanol and store at room temperature.
Kit 100% Ethanol to be Added
M2551-00 20 mL
M2551-01 80 mL
Preparing Reagents

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All of the Mag-Bind® FFPE RNA 96 Kit components are guaranteed for at least 12 months
from the date of purchase when stored as follows. Mag-Bind® DNase I and DNase I
Digestion Buer should be stored at -20°C. Mag-Bind® Particles SC and LPA Buer
should be stored at 2-8°C for long-term use. 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. All remaining components should be stored at room temperature. During shipment
or storage in cool ambient conditions, precipitates may form in RML Buer and MFB
Buer. Dissolve such deposits by warming the solution at 37°C and gently shaking.
Storage and Stability

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M2551 Mag-Bind® FFPE RNA 96 Kit Protocol
Mag-Bind® FFPE RNA 96 Kit Protocol - 96-well plates
Materials and Equipment to be Supplied by User:
• Nuclease-free 1.2 mL round-well plates
• Nuclease-free microplates
• Centrifuge with swing-bucket rotor capable of 4,000 x g
• RNase-free lter pipette tips
• Magnetic separation device for 96-well plates (MSD-01B or MSD-01)
• Water bath or heat block capable of 55°C
• Water bath or heat block capable of 80°C
• Water bath or heat block capable of 37°C
• 100% ethanol
• Sealing lm
Before Starting:
• Prepare RNA Wash Buer II and GFC Buer according to the Preparing Reagents
section on Page 4
• Set water bath or heat block to 55°C
• Set water bath or heat block to 80°C
• Set water bath or heat block to 37°C
1. Add 250 μL RML Buer into each well of a 1.2 mL round-well plate.
2. Cut 2-5 paran sample sections between 5-10 μm to be placed in each well of the 96
plate. Note: Do not use the rst 2-3 sections.
3. Immediately place 2-5 sections into each well of the round-well plate.
4. Centrifuge at 4,000 x gat room temperature for 2 minutes.
5. Incubate at 80°C for 15 minutes to melt the paran. Mix the sample a few times by
gently shaking the tube. Make sure that the tissue sections stay submerged in the
solution.
Note: Seal the plate with sealing lm to prevent evaporation during incubation.

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6. Add 25 µL Proteinase K Solution (20 mg/mL). 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.
7. Incubate at 80°C for 15 minutes.
8. Immediately centrifuge at 4,000 x gfor 5 minutes. The paran will form a thin layer
on top of the lysate solution.
9. Use a 1 mL pipette tip or large orice tip to penetrate the paran layer, transfer 200
μL cleared lysate into a new round-well plate.
10. Add 200 µL MFB Buer, 20 µL Mag-Bind® Particles SC, and 430 µL of 100% ethanol.
Mix thoroughly by vortexing for 20 seconds or pipetting up and down 10-20 times.
Note: If the RNA content from sample is expected low or miRNA is the target, then
add 10 µL LPA Buer.
11. Let sit at room temperature for 5-10 minutes.
12. Place the plate onto a magnetic separation device for deep-well plates and wait 7-10
minutes or until the Mag-Bind® Particles SC are cleared from solution.
Note: If using the MSD-01 magnetic separation device, a 500 µL processing 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 whole sample.
13. Aspirate and discard the cleared supernatant.
14. Remove the plate from the magnetic separation device.
15. Add 400 µL RNA Wash Buer II. Resuspend the Mag-Bind® Particles SC thoroughly by
vortexing for 20 seconds or pipetting up and down 10-20 times.
Note: RNA Wash Buer II must be diluted with 100% ethanol prior to use. Please see
instructions on Page 4.
M2551 Mag-Bind® FFPE RNA 96 Kit Protocol

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16. Place the plate onto the magnet separation device to magnetize the Mag-Bind®
Particles SC. Wait 3-5 minutes or until all the Mag-Bind® Particles SC are cleared from
solution.
17. Aspirate and discard the cleared supernatant. Remove any liquid drops from each
well.
18. Add 73.5 µL DNase I Digestion Buer and 1.5 µL RNase-free Mag-Bind® DNase I.
Resuspend the Mag-Bind® Particles SC thoroughly by vortexing for 20 seconds or
pipetting up and down 10-20 times.
19. Incubate at 37°C for 15 minutes.
20. Add 225 µL GFC Buer. Mix thoroughly by vortexing for 20 seconds or pipetting up
and down 10-20 times.
Note: GFC Buer must be diluted with 100% ethanol prior to use. Please see
instructions on Page 4.
21. Let sit at room temperature for 3-5 minutes.
22. Place the plate onto a magnet separation device to magnetize the Mag-Bind®
Particles SC. Wait 3-5 minutes or until all the Mag-Bind® Particles SC are cleared from
solution.
23. Aspirate and discard the cleared supernatant.
24. Remove the plate from the magnetic separation device.
25. Add 400 µL RNA Wash Buer II. Resuspend the Mag-Bind® Particles SC thoroughly by
vortexing for 20 seconds or pipetting up and down 10-20 times.
26. Place the plate onto a magnet separation device to magnetize the Mag-Bind®
Particles SC. Wait 3-5 minutes or until all the Mag-Bind® Particles SC are cleared from
solution.
M2551 Mag-Bind® FFPE RNA 96 Kit Protocol

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27. Aspirate and discard the cleared supernatant. Remove any liquid drops from tube.
28. Repeat Step 24-27 for a second RNA Wash Buer II wash step.
29. Air dry the Mag-Bind® Particles SC by leaving the plate on the magnetic separation
device for 10 minutes. Remove any residual liquid with a pipette.
30. Add 30-50 µL DEPC Water. Resuspend the Mag-Bind® Particles SC thoroughly by
vortexing for 30 seconds or pipetting up and down 30 times.
31. Let sit at room temperature for 10 minutes.
32. Place the plate onto a magnet separation device to magnetize the Mag-Bind®
Particles SC. Wait 3-5 minutes or until all the Mag-Bind® Particles SC are cleared from
solution.
33. Transfer the cleared supernatant containing puried RNA into a new nuclease-free
96-well microplate (not supplied) and seal with sealing lm.
34. Store the puried RNA at -80°C.
M2551 Mag-Bind® FFPE RNA 96 Kit Protocol

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M2551 Mag-Bind® FFPE RNA 96 Kit Protocol with Xylene
Mag-Bind® FFPE RNA 96 Kit Protocol with Xylene - 96-well
plates
Note: The following protocol uses xylene to remove paran from the FFPE sample.
Use fume hood and take proper protection during xylene extraction.
Materials and Equipment to be Supplied by User:
• 100% ethanol
• Xylene
• Nuclease-free 1.2 mL round-well plates
• Nuclease-free microplates
• Centrifuge with swing-bucket rotor capable of 4,000 x g
• RNase-free lter pipette tips
• Magnetic separation device for 96-well plates (MSD-01B or MSD-01)
• Water bath or heat block capable of 55°C
• Water bath or heat block capable of 80°C
• Water bath or heat block capable of 37°C
• Sealing lm
Before Starting:
• Prepare RNA Wash Buer II and GFC Buer according to the Preparing Reagents
section on Page 4
• Set water bath or heat block to 55°C
• Set water bath or heat block to 80°C
• Set water bath or heat block to 37°C
1. Add 1 mL xylene into each well of a 1.2 mL round-well plate.
2. Cut 2-5 paran sample sections between 5-10 μm.
Note: Do not use the rst 2-3 sections.
3. Immediately place 2-5 sections each well of the 1.2 mL round-well plate.
4. Let sit at room temperature for 2 minutes.
5. Mix thoroughly by vortexing for 20 seconds.

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6. Centrifuge at 4,000 x gat room temperature for 5 minutes to pellet the tissue.
Note: If the tissue does not form a tight pellet, centrifuge for an additional 3 minutes.
7. Carefully remove and discard the xylene without disturbing the pellets.
8. Add 1 mL 100% ethanol to each well. Mix thoroughly by vortexing for 20 seconds.
9. Centrifuge at 4,000 x gfor 5 minutes to pellet the tissue samples. The pellets should
appear opaque.
10. Carefully remove and discard the ethanol. 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 Proteinase K digestion
step. Ethanol residue will eect the eciency of the Proteinase K digestion. If a
vacuum oven is available, place the tube into the vacuum oven preset at 45°C for
10-30 minutes.
13. Add 250 μL RML Buer and 25 μL Proteinase K Solution (20 mg/mL). Resuspend the
pellet by vortexing or pipetting up and down 20 times.
14. Incubate at 55°C for 15 minutes.
15. Incubate at 80°C for 15 minutes.
16. Centrifuge at 4,000 x gat room temperature for 5 minutes.
17. Carefully transfer 200 μL cleared supernatant into a new round-well plate.
Mag-Bind® FFPE RNA 96 Protocol with Xylene

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18. Add 200 µL MFB Buer, 20 µL Mag-Bind® Particles SC, and 430 µL 100% ethanol. Mix
thoroughly by vortexing or pipetting up and down 10-20 times.
Note: If the RNA content from sample is expected low or miRNA is the target, add
10 µL LPA Buer.
19. Let sit at room temperature for 5-10 minutes.
20. Place the plate onto a magnetic separation device for deep-well plates and wait 7-10
minutes or until the Mag-Bind® Particles SC are cleared from solution.
Note: If using the MSD-01 magnetic separation device, a 500 µL processing 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 whole sample.
21. Aspirate and discard the cleared supernatant.
22. Remove the plate from the magnetic separation device.
23. Add 400 µL RNA Wash Buer II. Resuspend the Mag-Bind® Particles SC thoroughly by
vortexing for 20 seconds or pipetting up and down 10-20 times.
Note: RNA Wash Buer II must be diluted with 100% ethanol prior to use. Please see
instructions on Page 4.
24. Place the plate onto the magnet separation device to magnetize the Mag-Bind®
Particles SC. Wait 3-5 minutes or until all the Mag-Bind® Particles SC are cleared from
solution.
25. Aspirate and discard the cleared supernatant. Remove any liquid drops from each
well.
26. Add 73.5 µL DNase I Digestion Buer and 1.5 µL RNase-free Mag-Bind® DNase I.
Resuspend the Mag-Bind® Particles SC thoroughly by vortexing for 20 seconds or
pipetting up and down 10-20 times.
Mag-Bind® FFPE RNA 96 Protocol with Xylene

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27. Incubate at 37°C for 15 minutes.
28. Add 225 µL GFC Buer. Mix thoroughly by vortexing for 20 seconds or pipetting up
and down 10-20 times.
Note: GFC Buer must be diluted with 100% ethanol prior to use. Please see
instructions on Page 4.
29. Let sit at room temperature for 3-5 minutes.
30. Place the plate onto a magnet separation device to magnetize the Mag-Bind®
Particles SC. Wait 3-5 minutes or until all the Mag-Bind® Particles SC are cleared from
solution.
31. Aspirate and discard the cleared supernatant.
32. Remove the plate from the magnetic separation device.
33. Add 400 µL RNA Wash Buer II. Resuspend the Mag-Bind® Particles SC thoroughly by
vortexing for 20 seconds or pipetting up and down 10-20 times.
34. Place the plate onto a magnet separation device to magnetize the Mag-Bind®
Particles SC. Wait 3-5 minutes or until all the Mag-Bind® Particles SC are cleared from
solution.
35. Aspirate and discard the cleared supernatant. Remove any liquid drops from the
wells.
36. Repeat Step 32-35 for a second RNA Wash Buer II wash step.
37. Air dry the Mag-Bind® Particles SC by leaving the plate on the magnetic separation
device for 10 minutes. Remove any residual liquid with a pipette.
Mag-Bind® FFPE RNA 96 Protocol with Xylene

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38. Add 30-50 µL DEPC Water. Resuspend the Mag-Bind® Particles SC thoroughly by
vortexing for 30 seconds or pipetting up and down 30 times.
39. Let sit at room temperature for 10 minutes.
40. Place the plate onto a magnet separation device to magnetize the Mag-Bind®
Particles SC. Wait 3-5 minutes or until all the Mag-Bind® Particles SC are cleared from
solution.
41. Transfer the cleared supernatant containing puried RNA into a new nuclease-free
96-well microplate (not supplied) and seal with sealing lm.
42. Store the puried RNA at -80°C.
Mag-Bind® FFPE RNA 96 Protocol with Xylene

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Troubleshooting Guide
Please use this guide to troubleshoot any problems that may arise. For further assistance,
please contact our technical support sta, toll free, at 800-832-8896.
Possible Problems and Suggestions
Troubleshooting Guide
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
Make sure the sample is properly
stored and make sure the samples
are processed immediately after
collection or removal from storage.
RNA Wash Buer II was not
prepared correctly
Prepare RNA Wash Buer II by adding
ethanol according to the instructions.
Loss of magnetic beads
during operation Increase the beads collection time.
GFC Buer not diluted with
ethanol
Prepare GFC Buer as instructed on
the label.
RNA Wash Buer II was not
prepared correctly
Prepare RNA Wash Buer II by adding
ethanol according to the instructions.
Problem with
downstream
application
Degraded RNA During incubation at 37°C, do not
incubate sample over 15 minutes.
Carryover of the
magnetic beads
in the elution
Carryover the magnetic
beads in the eluted RNA
will not eect 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 plate.

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Notes: