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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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Summary of Contents for M2551-00

Page 1: ...Mag Bind FFPE RNA 96 Kit M2551 00 1 x 96 preps M2551 01 4 x 96 preps March 2018 ...

Page 2: ......

Page 3: ... 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 ...

Page 4: ...stion After two wash steps purified RNA is eluted with RNase free 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 sampl...

Page 5: ... 8 4 mL RML Buffer 35 mL 140 mL MFB Buffer 20 mL 80 mL GFC Buffer 10 mL 40 mL RNA Wash Buffer II 25 mL 2 x 50 mL LPA Buffer 1 1 mL 4 4 mL DNase I Digestion Buffer 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 ...

Page 6: ...nimize RNA degradation Wear gloves protective goggles and take great care when working with chemicals 1 Dilute RNA Wash Buffer 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 Buffer with 100 ethanol and store at room temperature Kit 100 Ethanol to be Added M2551 00 20 mL M2551 01 80 mL Preparing Reagents ...

Page 7: ...r 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 Buffer and MFB Buffer Dissolve such deposits by warming the solution at 37...

Page 8: ...uffer 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 Buffer into each well of a 1 2 mL round well plate 2 Cut 2 5 paraffin sample sections between 5 10 μm to be placed in each well of the 96 plate Note Do not use the first 2 3 sections 3 Immediately place 2 5 secti...

Page 9: ...then add 10 µL LPA Buffer 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 i...

Page 10: ... 20 times Note GFC Buffer 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 th...

Page 11: ...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 purified RNA into a new nuclease fre...

Page 12: ...or 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 film Before Starting Prepare RNA Wash Buffer II and GFC Buffer 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...

Page 13: ...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 effect the efficiency 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 Buffer and 25 μL Proteinase K Solution ...

Page 14: ...e 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 Buffer II Resuspend the Mag Bind Particles SC thoroughly by vortexing for 20 seconds or pipetting up and down 10 20 times Note RNA Wash Buffer II must be diluted with 100 ethanol prior to use Please see instructions on Page 4 ...

Page 15: ...Remove the plate from the magnetic separation device 33 Add 400 µL RNA Wash Buffer 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 cl...

Page 16: ... 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 purified RNA into a new nuclease free 96 well microplate not supplied and seal with sealing film 42 Store the purified RNA at 80 C Mag Bind FFPE RNA 96 Protocol with Xylene ...

Page 17: ...s not prepared correctly Prepare RNA Wash Buffer II by adding ethanol according to the instructions Loss of magnetic beads during operation Increase the beads collection time GFC Buffer not diluted with ethanol Prepare GFC Buffer as instructed on the label RNA Wash Buffer II was not prepared correctly Prepare RNA Wash Buffer II by adding ethanol according to the instructions Problem with downstrea...

Page 18: ...16 Notes ...

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