FFPE RNA Purification 96-Well Kit - Protocol
Contents
1. Cut sections up to 20 um thick from the interior of an FFPE tissue block using a microtome Trim off any excess paraffin Note Alternatively cut out up to 25 mg of unsectioned core from an FFPE block Trim off any excess paraffin Grind the sample into fine powder using liquid nitrogen Transfer the sections or ground block into the wells of the provided 96 Well Incubation Plate Add 1 mL of xylene to each sample Seal the wells with the provided adhesive tape Mix the sample by slight agitation Incubate at 55 C for 5 minutes Centrifuge the plate at 3 000 x g 3 000 RPM for 2 minutes Carefully remove the xylene from each well without dislodging the pellet Add 1 mL of 95 100 ethanol Mix by vortexing Centrifuge the sample at 3 000 x g 3 000 RPM for 2 minutes Carefully remove the ethanol without dislodging the pellet Repeat Step 1Ag to Step 1Aji for a second time Air dry the pellet for about 10 minutes at room temperature Note It is important to remove the ethanol completely Proceed to Step 2 Lysate Preparation Deparaffinization using microcentrifuge tubes a aro 2 0 6 a Cut sections up to 20 um thick from the interior of an FFPE tissue block using a microtome Trim off any excess paraffin Note Alternatively from an FFPE block cut out up to 25 mg of unsectioned core Trim off any excess paraffin Grind the sample into fine powder using liquid nitrogen Transfer the sections or ground bl2. dry the plate f Turn off vacuum and ventilate the manifold 5 RNA Elution a Replace the collection waste tray in the vacuum manifold with the provided 96 Well Elution Plate Complete the vacuum manifold assembly with the 96 Well Filter Plate b Add 75 uL of Elution Solution to each well of the plate c Apply vacuum for 2 minutes 6 Storage of RNA Use the provided adhesive tape to seal the 96 Well Elution Plate The purified RNA samples may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage B FFPE RNA Purification Using Centrifugation 3 Binding RNA to 96 Well Filter Plate a Place the 96 Well Filter Plate on top of a provided 96 Well Collection Plate b Apply up to 500 uL of the lysate with the ethanol from Step 2 into each well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes Note Depending on the starting material a small quantity of precipitates may appear in the lysate ethanol mix No additional step is required to remove these precipitates prior to application to the wells c Discard the flowthrough Reassemble the the 96 Well Filter Plate and the bottom plate Note Ensure that all of the lysate from each well has passed through into the bottom plate If the entire lysate volume has not passed centrifuge for an additional 2 minutes Optional Step Norgen s FFPE RNA Purification 96 Well K
3. FFPE RNA Purification Kit For All Protocols e For Vacuum Format Purification o Vacuum manifold with vacuum pump capable of generating a minimum pressure of 650 mbar or 25 in Hg such as Whatman UniVac 3 Vacuum to Collect Manifold o Sealing tape or pads e For 96 Well Centrifuge Format Purification o Centrifuge with rotor for 96 well plate assembly such as Thermo Fisher IEC Centra CL3 series or Beckman GS 15R 95 100 ethanol Xylene histological grade Incubators set at 55 C and 80 C B mercaptoethanol optional RNase Free Microcentrifuge Tubes optional Collection Waste Tray for vacuum manifold or 96 well bottom plate single or 96 well format for centrifugation Two 96 Well Collection Plates are provided with the kit Flowchart Procedure for Purifying Total RNA using Norgen s FFPE RNA Purification 96 Well Kit FFPE Tissue Samples Deparaffinization with xylene Wash with ethanol Add Digestion Buffer Proteinase K Incubate Add Binding Solution Ethanol Bind RNA Wash RNA Elute RNA Purified Total RNA Working with RNA RNases are very stable and robust enzymes that degrade RNA Autoclaving solutions and glassware is not always sufficient to actively remove these enzymes The first step when preparing to work with RNA is to create an RNase free environment The following precautions are recommended as your best defense against these enzymes e The RNA area should be located away from
4. incubation time Do not exceed the recommended amounts of starting materials The amount of starting material may need to be decreased if the well shows clogging below the recommended levels See also Clogged Wells below It is recommended that the Elution Solution supplied with this kit be used for maximum RNA recovery Ensure that the appropriate amount of ethanol and Binding Solution are added to the lysate before binding to the well Ensure that 50 mL of 95 ethanol is added to the supplied Wash Solution prior to use Different tissues and cells have different RNA contents and thus the expected yield of RNA will vary greatly from these different sources Please check literature to determine the expected RNA content of your starting material RNA is Degraded Insufficient solubilization of cells or tissues Insufficient Vacuum Maximum number of cells or amount of tissue exceeds kit specifications Clarified lysate was not used for the binding step Centrifuge temperature too low FFPE sample is old RNase contamination Procedure not performed quickly enough Improper storage of the purified RNA Prolonged incubation at 80 C Starting material may have a high RNase content Possible Cause Solution and Explanation Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue Ensure that a vacuum pressure of at least 650 mbar or 25 in Hg is develope
5. recommended that B mercaptoethanol be added to the Binding Solution 10 Possible Cause Solution and Explanation RNA was not Traces of salt from the binding step may remain in the washed 3 times sample if the well is not washed 3 times with Wash with the provided Solution Salt may interfere with downstream Wash Solution applications and thus must be washed from the well RNA does not perform well in Ethanol carryover downstream applications Ensure that the dry spin under the Well Wash procedure is performed in order to remove traces of ethanol prior to elution Ethanol is known to interfere with many downstream applications Formalin crosslink Ensure the sufficient incubation at 80 C is performed in was not completely Step 2b Do not exceed 15 minutes of incubation at 80 C reversed as this will increase RNA fragmentation Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2012 Norgen Biotek Corp P125400 4 1
6. Fax 905 227 1061 BIOTEK wi CORPORATION Email techsupport norgenbiotek com gt k 3430 Schmon Parkway N R E N Thorold ON Canada L2V 4Y6 gt Phone 866 667 4362 e 905 227 8848 FFPE RNA Purification 96 Well Kit Product Insert Product 25400 Norgen s FFPE RNA Purification Kit provides a rapid method for high throughput isolation and purification of total RNA including microRNA from formalin fixed paraffin embedded FFPE tissue samples Using formalin to fix tissues leads to crosslinking of the RNA and proteins and the process of embedding the tissue samples can also lead to fragmentation of the RNA over time Norgen s FFPE RNA Purification Kit provides conditions that allow for the partial reversing of the formalin modifications resulting in a high quality and yield of RNA The kit is able to purify all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA depending on the age of the FFPE tissue as the degree of fragmentation of the RNA will increase over time The RNA is preferentially purified from other cellular components without the use of phenol or chloroform The purified RNA is of the highest integrity and can be used in a number of downstream applications including qRT PCR reverse transcription PCR primer extension expression array assays and microarray analyses Norgen s Purification Technology Purification is based on 96 well column chromatography using No
7. ach well has passed through into the collection waste tray If the entire lysate volume has not passed apply vacuum for an additional 2 minutes Optional Step Norgen s FFPE RNA Purification 96 Well Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional On Plate DNA Removal Protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications This step should be performed at this point in the protocol 4 RNA Wash a Apply 400 uL of Wash Solution to each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes Note Ensure the entire wash solution has passed through into the collection waste tray by inspecting the 96 Well Filter Plate If the entire wash volume has not passed apply vacuum for an additional 2 minutes b Turn off vacuum and ventilate the manifold Discard the flowthrough c Reassemble the 96 Well Filter Plate and the vacuum manifold Repeat steps 4a and 4b to wash column for a second time d Reassemble the 96 Well Filter Plate and the vacuum manifold Repeat steps 4a and 4b to wash column for a third time e Pat the bottom of the 96 Well Filter Plate dry Reassemble the 96 Well Filter Plate and the vacuum manifold Apply vacuum for an additional 5 minutes in order to completely
8. d Refer to specifications to determine if amount of starting material falls within kit specifications Ensure that after the lysis step the sample is centrifuged if a significant amount of debris is present and that only the clarified lysate is used in subsequent steps Ensure that the centrifuge remains at room temperature throughout the procedure Temperatures below 15 C may cause precipitates to form that can cause the wells to clog The quality of RNA purified may drastically decrease in old samples For best performance freshly prepared samples are highly recommended RNases may be introduced during the use of the kit Ensure proper procedures are followed when working with RNA Please refer to Working with RNA at the beginning of this user guide In order to maintain the integrity of the RNA it is important that the procedure be performed quickly This is especially important for the Cell Lysate Preparation Step in the Animal Tissue protocol since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized For short term storage RNA samples may be stored at 20 C for a few days It is recommended that samples be stored at 70 C for longer term storage In order to reverse formalin crosslinks an incubation at 80 C is required Do not exceed 15 minutes of incubation at 80 C as this will increase RNA fragmentation For starting materials with high RNAase content it is
9. d at 20 C for a few days Itis recommended that samples be placed at 70 C for long term storage Appendix A Protocol for Optional On Column DNA Removal Norgen s FFPE RNA Purification 96 Well Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional protocol is provided below for maximum removal of residual DNA that may affect sensitive downstream applications It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step 1 For every on column reaction to be performed prepare a mix of 5 uL of DNase and 75 uL of Enzyme Incubation Buffer using Norgen s RNase Free DNase Kit Product 25710 Mix gently by inverting the tube a few times DO NOT VORTEX Note If using an alternative DNase I prepare a working stock of 0 25 Kunitz unit wL RNase free DNase solution according to the manufacturer s instructions A 75 uL aliquot is required for each well to be treated 2 Perform the appropriate FFPE RNA Isolation Procedure up to and including Binding RNA to 96 Well Filter Plate Steps 1 to 3 of all protocols 3 For Vacuum Manifold Apply 400 uL of Wash Solution to each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or a pad provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes For Centrifugation Apply 400 uL of Wash Solution to each well of the 96 Wel
10. ecular biology grade water aliquot into small fractions and store the unused portions at 20 C until needed e Prepare a working concentration of the Wash Solution by adding 90 mL of 95 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 120 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Optional For sensitive downstream applications or target transcripts of low quantity add 10 uL of B mercaptoethanol provided by the user to each 1 mL of Binding Solution required B mercaptoethanol is toxic and should be dispensed in a fume hood Alternatively the Binding Solution can be used as provided e The maximum recommended input is five sections of lt 20 um thick Alternatively an unsectioned block of up to 25 mg may be used e It is important to obtain sections from the interior of an FFPE block in order to minimize RNA damage by oxidation e It is important to work quickly during this procedure Section 1 FFPE Tissue Deparaffinization and Lysate Preparation Note Deep well 96 Well Incubation Plates are provided with the kit for high throughput preparation of lysate from FFPE samples Step 1A Alternatively the user may use RNase free microcentrifuge tubes not provided for lysate preparation Step 1B 1A Deparaffinization using Provided 96 Well Incubation Plate 1B a sa 79 oO a
11. it isolates total RNA with minimal amounts of genomic DNA contamination However an optional On Plate DNA Removal Protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications This step should be performed at this point in the protocol 4 RNA Wash a Apply 400 uL of Wash Solution to each well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes Note Ensure the entire wash solution has passed through into the bottom plate by inspecting the 96 Well Filter Plate If the entire wash volume has not passed centrifuge for an additional 2 minutes Discard the flowthrough Reassemble the 96 Well Filter Plate and the bottom plate Repeat steps 4a and 4b to wash column for a second time Repeat steps 4a and 4b to wash column for a third time Pat the bottom of the 96 Well Filter Plate dry Reassemble the 96 Well Filter Plate and the bottom plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 5 minutes in order to completely dry the plate vaoo 5 RNA Elution a Stack the 96 Well Filter Plate on top of the 96 Well Elution Plate b Add 75 uL of Elution Solution to each well of the 96 Well Filter Plate c Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes 6 Storage of RNA Use the provided adhesive tape to seal the 96 Well Elution Plate The purified RNA sample may be store
12. l Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes 4 Discard the flowthrough Reassemble the 96 Well Filter Plate with the vacuum manifold or the bottom plate 5 Apply 75 uL of the RNase free DNase solution prepared in Step 1 to each well of the 96 Well Filter Plate For Vacuum Manifold Apply vacuum for 30 seconds For Centrifugation Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 30 seconds 6 After the centrifugation or vacuum in Step 5 pipette the flowthrough that is present in the collection plate back onto the top of the well Note Ensure Step 6 is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species 7 Incubate the assembly at 25 30 C for 15 minutes 8 Without any further centrifugation proceed directly to RNA Wash Section 2A Step 4c for Vacuum Manifold procedure or Section 2B Step 4c for Centrifugation procedure Troubleshooting Guide Problem Poor RNA Recovery Incomplete lysis of cells or tissue Wells has become clogged An alternative elution solution was used Ethanol or Binding Solution was not added to the lysate Ethanol was not added to the Wash Solution Low RNA content in cells or tissues used Possible Cause Solution and Explanation Ensure that the appropriate amount of Digestion Buffer with Proteinase K added was used Increase the
13. microbiological work stations e Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc It is recommended that gloves are changed frequently to avoid contamination e There should be designated solutions tips tubes lab coats pipettes etc for RNA only e All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water e Clean all surfaces with commercially available RNase decontamination solutions e When working with purified RNA samples ensure that they remain on ice during downstream applications Procedures For Vacuum Manifold All vacuum steps are performed at room temperature The correct pressure can be calculated using the conversions 1 mbar 100 Pa 0 0394 in Hg 0 75 mm Hg 0 0145 psi For Centrifugation All centrifugation steps are performed at room temperature The correct rom can be calculated using the formula RPM RCF 1 118 x 105 r where RCF required gravitational acceleration relative centrifugal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force Notes Prior to Use e Ensure that all solutions are at room temperature prior to use e All enzymes provided should remain at the storage temperature indicated on each vial until use e Reconstitute the Proteinase K in 1 mL of mol
14. ock into an RNase free microcentrifuge tube Add 1 mL of xylene to the sample Mix by vortexing Incubate at 55 C for 5 minutes Centrifuge the sample at 14 000 x g 14 000 RPM for 2 minutes Carefully remove the xylene without dislodging the pellet Add 1 mL of 95 100 ethanol Mix by vortexing Centrifuge the sample at 14 000 x g 14 000 RPM for 2 minutes Carefully remove the ethanol without dislodging the pellet Repeat Step 1Bg to Step 1Bi for a second time Air dry the pellet for about 10 minutes at room temperature Note It is important to remove the ethanol completely Proceed to Step 2 Lysate Preparation 2 Lysate Preparation a Add 200 uL of Digestion Buffer and 10 uL of the reconstituted Proteinase K to the sample Seal the plate with adhesive tape or close the cap of the microcentrifuge tube Mix by agitation for samples in the 96 Well Incubation Plate or vortexing for those in microcentrifuge tubes b Incubate at 55 C for 15 minutes followed by 80 C for 15 minutes Agitate or vortex to mix occasionally for 96 Well Incubation Plate or microcentrifuge respectively Note Do not exceed 15 minutes of incubation at 80 C as this will increase RNA fragmentation Note For the 96 Well Incubation Plate gently tap the plate against the bench top after the incubation to remove any condensation on top of the wells c Add 200 uL of Binding Solution Agitate or vortex to mix for 96 Well Incubation Plate or microcen
15. on e High yields of total RNA e Isolate total RNA from large rRNA down to microRNA miRNA e No phenol or chloroform extractions Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature The DNAse should be stored at 20 C upon arrival The Proteinase K should be stored at 20 C upon arrival and after reconstitution These reagents should remain stable for at least 1 year in their unopened containers Kit Components Proteinase K DNase Adhesive Tae o sn Precautions and Disclaimers This kit is designed for research purposes only Itis not intended for human or diagnostic use Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with whole blood The Binding Solution contains guanidine salts and should be handled with care Guanidine salt forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Customer Supplied Reagents and Equipment You must have the following in order to use the
16. rgen s proprietary resin as the separation matrix The RNA is preferentially purified from other cellular components without the use of phenol or chloroform The process first involves deparaffinization of the FFPE samples through a series of xylene and ethanol washes Next the FFPE samples are digested with the provided Proteinase K and Digestion Buffer Binding Solution and ethanol are then added to the lysate and the solution is loaded onto the 96 Well Filter Plate for purification The purification could be performed on either a vacuum manifold or using centrifugation Norgen s resin binds nucleic acids in a manner that depends on ionic concentrations thus only the RNA will bind to the resin while other contaminants will be removed in the flowthrough or retained on the top of the resin At this point any remaining traces of genomic DNA can be digested using an optional protocol allowing for pure RNA samples to be isolated The bound RNA is then washed with the provided RNA Wash Solution in order to remove any impurities and the purified total RNA is eluted with the Elution Solution Specifications Kit Specifications Maximum Binding Capacity per Well 50 ug Maximum Loading Volume per Well 500 uL Size of RNA Purified All sizes including small RNA lt 200 nt 5 sections lt 20 uM thick Maximum Amount of Starting Material 25 mg of unsectioned block Advantages e Fast and easy processing using either a vacuum manifold or centrifugati
17. trifuge respectively d Add 400 uL of 95 100 ethanol Agitate or vortex to mix for 96 Well Incubation Plate or microcentrifuge respectively Section 2 FFPE RNA Purification Note The purification of total RNA from the lysate prepared in Section 1 could be performed using either a vacuum manifold or centrifugation For purification using vacuum please follow the procedure outlined in Section 2A For purification using centrifugation please follow the procedure outlined in Section 2B A FFPE RNA Purification Using Vacuum Manifold 3 Binding RNA to 96 Well Filter Plate a Assemble the 96 Well Filter Plate and the vacuum manifold according to manufacturer s recommendations Note The provided 96 Well Collection Plate can be used as the collection waste tray if desired b Apply the lysate with the ethanol from Step 2 into each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes Note Depending on the starting material a small quantity of precipitates may appear in the lysate ethanol mix No additional step is required to remove these precipitates prior to application of the mixture to the wells c Turn off vacuum and ventilate the manifold Discard the flowthrough Reassemble the 96 Well Filter Plate and the vacuum manifold Note Ensure that all of the lysate from e
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