54300 - Protocol (50 prep)
Contents
1. 250 uL of 96 100 ethanol Vortex to mix 8 Binding DNA to Column a Assemble an DNA Purification Micro Column with one of the provided collection tubes b Apply up to 600 uL of the lysate with the ethanol from Step 7f onto the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube Repeat Step 8b and 8c until all lysate has passed through the column a9 9 DNA Column Wash a Apply 600 uL of Wash Solution A to the column and centrifuge for 1 minute at 14 000 x g 14 000 RPM Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute g Discard the flowthrough and reassemble the spin column with its collection tube Apply 600 uL of Wash Solution A to the column and centrifuge for 1 minute at 14 000 xg 14 000 RPM h Discard the flowthrough and reassemble the spin column with its collection tube i Wash column a third time by adding another 600 uL of Wash Solution A and centrifuging for 1 minute at 14 000 x g 14 000 RPM j Discard the flowthrough and reassemble the spin column with its collection tube k Spin the column for 2 minutes 14 000 x g 14 000 RPM in order to thoroughly dry the resin Discard the collection tube 10 DNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the2. gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com FFPE RNA DNA Purification Plus Kit Product Insert Product 54300 Norgen s FFPE RNA DNA Purification Plus Kit provides a rapid method for the sequential isolation and purification of total RNA including microRNA and genomic DNA from formalin fixed paraffin embedded FFPE tissue samples Using formalin to fix tissues leads to crosslinking of the nucleic acids and proteins and the process of embedding the tissue samples can also lead to fragmentation of the nucleic acids over time Norgen s FFPE RNA DNA Purification Plus Kit provides conditions that allow for the partial reversing of the formalin modifications resulting in a high quality and yield of nucleic acids 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 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 real time PCR reverse transcription PCR Northern blotting RNase protection and primer extension and expression array assays The purified genomic DNA is also of
3. 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 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 All centrifugation steps are carried out in a benchtop microcentrifuge Various speeds are required for different steps so please check your microcentrifuge specifications to ensure that it is capable of the proper speeds All centrifugation steps are performed at room temperature The correct rom can be calculated using the formula RPM RCF 1 118 x 10 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 All centrifugation steps are carried out in a benchtop mic
4. d to the supplied Wash Solution A prior to use Solutions Different tissues and cells have different nucleic acid Low nucleic acid contents and thus the expected yield of nucleic acid will content in cells or vary greatly from these different sources Please check tissues used literature to determine the expected nucleic acid content of your starting material Insufficient Ensure that the appropriate amount of Digestion Buffer solubilization of A with Proteinase K added was used Increase the cells or tissues incubation time Maximum number Of sections or Refer to specifications to determine if amount of starting amount of tissue A iaa UTA material falls within kit specifications exceeds kit specifications Clogged Column Clarified lysate was not used for the binding step Ensure that after the lysis step the sample is centrifuged if significant precipitates are present and that only the clarified lysate is used in subsequent steps Centrifuge temperature too low Ensure that the centrifuge remains at room temperature throughout the procedure Temperatures below 15 C may cause precipitates to form that can cause the columns to clog Problem Possible Cause Solution and Explanation FFPE sample is old The quality of RNA purified may drastically decrease in old samples For best performance freshly prepared samples are highly recommended RNase contamination RNases may be introduced duri
5. e supplied in they can easily be identified as follows o DNA Purification Micro Columns Column has predominately white contents o RNA Purification Micro Columns Column has predominately black contents Section 1 Deparaffinization of FFPE Samples 1 Deparaffinization a ATT Ta 29 AaOt Cut four 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 10 mg of unsectioned core Trim off any excess paraffin Grind the sample into fine powder using liquid nitrogen Transfer the sections or ground block into an RNase free microcentrifuge tube Add 1 mL of xylene to the sample Mix by vortexing Incubate at 50 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 96 100 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 1g to Step 1i 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 Section 2 Total RNA Purification Section 2 Total RNA Purification 2 Lysate Preparation a b C Add 300 uL of Digestion Buffer A and 10 uL of the reconstituted Proteinase K to the sample Mix by vortex
6. ed spin at 14 000 x g for an additional minute After the centrifugation in Step b pipette the flowthrough that is present in the collection tube back onto the top of the column Note Ensure that Step c is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species Incubate at room temperature for 15 minutes Proceed to Step 4b without further centrifugation Note Add the Wash Solution A directly to the column containing the Enzyme Incubation Buffer A mix containing the RNase free DNase I Troubleshooting Guide Problem Possible Cause Solution and Explanation incompl te Was ek Ensure that the appropriate amount of Digestion Buffer POS y A and Proteinase K was used Increase the incubation cells or tissue time Do not exceed the recommended amounts of starting materials The amount of starting material may need to Column has i become clogged be decreased if the column shows clogging below the recommended levels See also Clogged Column below An alternative It is recommended that the Elution Solution A and elution solution was Elution Buffer F supplied with this kit be used for used maximum RNA or DNA recovery Poor RNA or DNA Recover 4 Eak lng Ensure that the appropriate amount of ethanol and Buffer RL is added to the lysate before binding to the column to the lysate ue Wess Ensure that 90 mL of 96 100 ethanol is adde
7. erosaink Ensure the sufficient incubation at 80 or 90 C is was not completely performed to remove formalin crosslink reversed Genomic Large amounts of Perform RNAse free DNasel digestion on the RNA DNA starting material sample after elution to remove genomic DNA contamination used contamination 10 Related Products Product RNA Protein Purification Kit 24100 RNA DNA Protein Purification Plus Kit 47700 FFPE RNA Purification Kit 25300 FFPE RNA DNA Purification Kit 25000 FFPE RNA Purification 96 Well Kit 25400 Cytoplasmic amp Nuclear RNA Purification Kit 21000 Leukocyte RNA Purification Kit 21200 Total RNA Purification Kit 17200 100b RNA Ladder 15002 1kb RNA Ladder 15003 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 2015 Norgen Biotek Corp P154300 4 M14 11
8. ing Incubate at 55 C for 15 minute Vortex to mix occasionally Allow the sample to cool down by placing the tube containing the sample on ice for 3 minutes Centrifuge the sample at 14 000 x g 14 000 RPM for 3 minutes Carefully transfer the RNA containing supernatant to a new an RNase free microcentrifuge tube Retain the microcentrifuge tube containing the pellet for DNA Purification Note The DNA containing pellet can be stored for 2 hours at room temperature for up 24 hours at 2 8 C or at 20 C for extensive storage Incubate the tube of the RNA containing lysate at 80 C for 15 minutes Vortex to mix occasionally Note Do not exceed 15 minutes of incubation at 80 C as this will increase RNA fragmentation g Add 300 uL of Buffer RL Vortex to mix h Add 600 uL of 96 100 ethanol Vortex to mix 3 Binding RNA to Column a Assemble an RNA Purification Micro Column with one of the provided collection tubes b Apply up to 600 uL of the lysate with the ethanol from Step 2h onto the column and centrifuge for 1 minute at 2 3 500 x g 6 000 RPM Note Ensure the entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional minute at 14 000 x g 14 000 RPM c Discard the flowthrough Reassemble the spin column with its collection tube d Repeat Step 3b and 3c until all lysate has passed through the column Opt
9. ional Step Norgen s FFPE RNA DNA Purification Plus Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional On Column 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 using the provided DNase I 4 RNA Column Wash a Apply 500 uL of Wash Solution A to the column and centrifuge for 1 minute at 14 000 x g 14 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute b Apply 500 uL of Wash Solution A to the column and centrifuge for 1 minute at 14 000 x g 14 000 RPM c Discard the flowthrough and reassemble the spin column with its collection tube d Wash column a third time by adding another 500 uL of Wash Solution A and centrifuging for 1 minute at 14 000 x g 14 000 RPM e Discard the flowthrough and reassemble the spin column with its collection tube Spin the column for 2 minutes 14 000 x g 14 000 RPM in order to thoroughly dry the resin Discard the collection tube n 5 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 20 50 uL of Elution Solution A to the column c Centrif
10. kit b Add 20 50 uL of Elution Buffer F to the column Incubate the assembly at room Cc temperature for 1 minute Centrifuge 1 minute at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute Note For maximum DNA recovery it is recommended that a second elution be performed into a separate microcentrifuge tube Repeat Steps 10b and 10c 11 Storage of DNA The purified DNA may be stored at 4 C for a few days It is recommended that samples be placed at 20 or 70 C for long term storage Appendix A Protocol for Optional On Column DNA Removal Notes Prior to Use This optional step is carried out if genomic DNA free RNA is required Prepare a DNase mixture by adding 4 uL of the provided RNase free DNase to 96 uL of Enzyme Incubation Buffer A for each isolation Apply 500 uL of Wash Solution A to the column and centrifuge at 14 000 x g 14 000 RPM for 2 minutes Discard the flowthrough Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute Apply 100 uL Enzyme Incubation Buffer A mix containing the RNase free DNase to the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute Note Ensure that the entire volume of DNase mix passes through the column If need
11. lumn Binding Capacity RNA 35 ug Column Binding Capacity gDNA 10 ug Maximum Column Loading Volume 600 uL All sizes including small RNA lt 200 nt 4 sections lt 20 uM thick 10 mg of unsectioned block Size of RNA Purified Maximum Amount of Starting Material Advantages e Fast and easy processing using rapid spin column format Separate fraction of RNA and DNA High yields and quality of nucleic acids Isolate total RNA from large rRNA down to microRNA miRNA No phenol or chloroform extractions Kit Components Component Product 54300 50 preps Digestion Buffer A 2x25 mL Buffer RL 40 mL Enzyme Incubation Buffer A 6 mL Wash Solution A 2 x 38 mL Elution Solution A 6 mL Elution Buffer F 15 mL Proteinase K 2x12mqg DNase 1 vial RNA Purification Micro Columns 50 DNA Purification Micro Columns 50 Collection Tubes 100 Elution Tubes 100 Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature The DNAse and Proteinase K should be stored at 20 C upon arrival These reagents should remain stable for at least 1 year in their unopened containers Precautions and Disclaimers This kit is designed for research purposes only It is 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 informatio
12. n please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com The Buffer RL contains guanidinium salts and should be handled with care Guanidinium salts form 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 FFPE RNA DNA Purification Plus Kit Benchtop microcentrifuge 50 55 C Incubator or Water Bath 80 C Incubator or Water Bath 90 C Incubator or Water Bath 96 100 ethanol Xylene histological grade Ice Bucket Flowchart Procedure for Purifying Total RNA and DNA using Norgen s FFPE RNA DNA Purification Plus Kit FFPE Tissue Samples oF Deparaffinization with xylene Wash with ethanol Add Digestion Buffer A Proteinase K Incubate SPIN 3 Supernatant Pellet Add Digestion Buffer A Proteinase K Incubate Add Buffer RL Add ethanol Incubate Add Buffer RL Add ethanol l l Bind RNA Bind DNA SPIN 4 SPIN 3 Wash RNA Wash DNA SPIN J SPIN 3 Elute RNA Elute DNA SPIN J SPIN Purified Total RNA Purified DNA 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
13. ng 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 RNA is Too long incubation important that the procedure be performed according to Degraded of lysate at high the time indicated This is especially important for the aka erat re g lysate preparation step when the sample is incubated at P 55 and 80 C for 15 minutes each Incubation beyond the time indicated may lead to fragmentation of RNA impt perstorade of For short term storage RNA samples may be stored at he id aa 20 C for a few days It is recommended that samples P be stored at 70 C for longer term storage Prolonged In order to reverse formalin crosslinks an incubation at incubation at 55 C 55 C is required which may lead to degraded RNA Nucleic acids were Traces of salt from the binding step may remain in the not washed 3 times sample if the column 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 column Nucleic acids does not Ensure that the dry spin under the Column Wash perform well procedure is performed in order to remove traces of in Elnanigh carryover ethanol prior to elution Ethanol is known to interfere with downstream many downstream applications applications Formalin
14. rocentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature e A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed 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 each vial of the Proteinase K in 600 uL of molecular biology grade water aliquot into small fractions and store the unused portions at 20 C until needed e Prepare a working concentration of each bottle of the Wash Solution A by adding 90 mL of 96 100 ethanol provided by the user to the supplied bottles containing the concentrated Wash Solution A This will give a final volume of 128 mL The label on the bottles has a box that may be checked to indicate that the ethanol has been added Wash Solution A is used for both RNA and DNA Purification e The maximum recommended input is four sections of lt 20 um thick Alternatively an unsectioned block of up to 10 mg may be used It is important to obtain sections from the interior of an FFPE block in order to minimize RNA or DNA damage by oxidation It is important to work quickly during this procedure This kit is provided with 2 separate columns When columns are removed from the labelled bags they ar
15. the highest quality and can be used in PCR reactions sequencing Southern blotting and SNP analysis Norgen s Purification Technology Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix The nucleic acids are 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 A using an incubation time which is specific for the recovery of RNA please see the flow chart on page 3 The soluble lysate containing the RNA is then collected for RNA purification while the remaining sample is further digested for DNA Buffer RL and ethanol are then added to the lysate containing RNA or DNA and the solution is loaded onto an RNA Purification Micro Column or a DNA Purification Micro Column respectively Norgen s resin binds nucleic acids in a manner that depends on ionic concentrations thus only the RNA or DNA will bind to the column while the contaminants will be removed in the flowthrough or retained on the top of the resin The bound nucleic acid is then washed with the provided Wash Solution in order to remove any impurities and the purified nucleic acid is eluted with the Elution Solution A or Elution Buffer F Specifications Kit Specifications Co
16. uge for 2 minutes at 200 x g 2 000 RPM followed by a 1 minute spin at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute Note As little as 15 uL of Elution Solution A could be used for higher RNA concentration However the RNA yield may be reduced when a smaller elution volume is used For maximum RNA recovery it is recommended that a second elution be performed into a separate microcentrifuge tube Repeat Steps 5b and 5c 6 Storage of RNA The purified RNA may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage Section 3 Total DNA Purification 7 Lysate Preparation a Add 300 uL of Digestion Buffer A and 10 uL of the reconstituted Proteinase K to the DNA containing pellet obtained from Step 2e Mix by vortexing b Incubate at 55 C for 1 hour Vortex to mix occasionally c Incubate at 90 C for 2 hour Vortex gently occasionally to mix d Allow the sample to cool down by placing the tube containing the sample on ice for 3 minutes Note Norgen s FFPE RNA DNA Purification Plus Kit isolates DNA with minimal amounts of RNA contamination However if it is desirable to remove any trace amount of RNA add 4 uL of RNase A 10 mg mL to the cooled lysate and incubate at room temperature for 5 minutes e Add 300 uL of Buffer RL Vortex to mix f Add
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