User Manual
Contents
1. Place the column into a fresh 1 7 mL Elution tube provided with the kit Add 20 50 uL of Elution Solution to the column Incubate the assembly at room temperature for 1 minute Centrifuge 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 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 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 for each isolation Apply 400 uL of Wash Solution to the column and centrifuge 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 of Enzyme Incubation Buffer mix containing the RNase free DNase to the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute Note2. Ensure that the entire volume of DNase mix passes through the column If needed 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 4c without further centrifugation 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 Troubleshooting Guide Possible Cause Solution and Explanation Poor RNA Recovery Clogged Column Incomplete lysis of cells or tissue Column 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 Insufficient solubilization of cells or tissues Maximum number of cells or amount of tissue exceeds kit specifications Clarified lysate was not used for the binding step Centrifuge temperature too low Ensure that the appropriate amount of Digestion Buffer with Protein
3. findings To minimize irregularities in diagnostic results suitable controls for downstream applications should be used Norgen s FFPE RNA Purification Kit Dx is intended for use by professional users such as technicians physicians and biologists experienced and trained in molecular biological techniques Norgen s FFPE RNA Purification Kit Dx does not provide a diagnostic result It is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay Kit Components Component Product 25300 50 preps Qt B or TREE S7 at fv Ca not Use ES Batch Catalogue Contains Manu In Vitro Consult Temper reuse Code Number sufficient facturer Diagnostic instructions ature for lt n gt Medical for use limitation tests Device Made in Canada Norgen Biotek Advantages e CE IVD marked in accordance with EU Directive 98 79 EC Fits into in vitro diagnostic workflows Fast and easy processing using rapid spin column format Isolate total RNA from large rRNA down to microRNA miRNA No phenol or chloroform extractions Specifications Kit Specifications Maximum Column Binding Capacity RNA Maximum Column Loading Volume 650 uL Size of RNA Purified All sizes including small RNA Sze of RNA Purea O lt 200 nt 5 sections lt 20 uM thick Maximum Amount of Starting Material a a ached oieck Storage Conditions and Product Stability The DNAse I and P
4. of salt from the binding step may remain in the sample if the column is not washed 3 times with Wash Solution Salt may interfere with downstream applications and thus must be washed from the column Ensure that the dry spin under the Column Wash procedure is performed in order to remove traces of ethanol prior to elution Ethanol is Known to interfere with many downstream applications Ensure the sufficient incubation at 80 C is performed in Step 2b Do not exceed 15 minutes of incubation at 80 C as this will increase RNA fragmentation Product Use Restriction Norgen s FFPE RNA Purification Kit Dx provides a rapid method for the isolation and purification of total RNA including microRNA from formalin fixed paraffin embedded FFPE tissue samples subsequent in vitro diagnostic use This kit is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA followed by signal detection or amplification Any diagnostic results generated using the RNA isolated with Norgen s FFPE RNA Purification Kit Dx in conjunction with an in vitro diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results suitable controls for downstream applications should be used Norgen s FFPE RNA Purification Kit Dx is intended for use by professional users such as technicians physicians and biologists experie
5. t SPIN 3 Purified Total RNA Notes Prior to Use All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature 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 Ensure that all solutions are at room temperature prior to use All enzymes provided should remain at the storage temperature indicated on each vial until use Reconstitute 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 Prepare a working concentration of the Wash Solution by adding 50 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 72 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added The maximum recommended input is five sections of lt 20 um thick Alternatively an unsectioned block of up to 25 mg may be used It is important to obtain sections from the interior of an FFPE block in order to minimize RNA damage by oxidation lt is important to work quickly during this procedure 1 Deparaffinization a ATH Tere e Cut sections up to 20 um thick from the interior of
6. am applications 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 RNA was not washed 3 times with the provided Wash Solution Ethanol carryover Formalin crosslink was not completely reversed 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 recommended that B mercaptoethanol be added to the Binding Solution Traces
7. 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 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 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 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 Step 2 Lysate Preparation 2 Lysate Preparation a b Add 300 uL of Digestion Buffer and 10 uL of the reconstituted Proteinase K to the sample Mix by vortexing Incubate at 55 C for 15 minutes followed by 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 Add 300 uL of Binding Solution Vortex to mix Add 600 uL of 96 100 ethanol Vortex to mix 3 Binding RNA to Column a b C d Assemble a column with one of the provided collection tubes Apply
8. ase K added was used Increase the incubation time Do not exceed the recommended amounts of starting materials The amount of starting material may need to be decreased if the column shows clogging below the recommended levels See also Clogged Column 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 column Ensure that 50 mL of 96 100 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 Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue 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 columns to clog Possible Cause Solution and Explanation RNA is Degraded RNA does not perform well In downstre
9. hR 3430 Schmon Parkway N QO E N Thorold ON Canada L2V 4Y6 Phone 905 227 8848 BIOTEK a CORPORATION Fax 905 227 1061 Email techsupport norgenbiotek com FFPE RNA Purification Kit Dx Product Insert Dx25300 CE AR PIDx25300 2 Intended Use Norgen s FFPE RNA Purification Kit Dx provides a rapid method for the isolation and purification of total RNA including microRNA from formalin fixed paraffin embedded FFPE tissue samples subsequent in vitro diagnostic use 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 Dx 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 This kit is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA followed by signal detection or amplification Any diagnostic results generated using the RNA isolated with Norgen s FFPE RNA Purification Kit Dx in conjunction with an in vitro diagnostic assay should be interpreted with regard to other clinical or laboratory
10. nced and trained in molecular biological techniques Norgen s FFPE RNA Purification Kit Dx does not provide a diagnostic result It is the sole responsibility of the user to use and validate the kit in conjunction with a downstream in vitro diagnostic assay The respective user is liable for any and all damages resulting from application of Norgen s FFPE RNA Purification Kit Dx for use deviating from the intended use as specified in the user manual All products sold by Norgen Biotek are subjected to extensive quality control procedures and are warranted to perform as described when used correctly Any problems should be reported immediately The kit contents are for laboratory use only and they must be stored in the laboratory and must not be used for purposes other than intended The kit contents are unfit for consumption Authorized Representative EMERGO EUROPE Molenstraat 15 2513 BH The Hague The Netherlands pm Norgen Biotek Corp 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 PIDx25300 2
11. roteinase K should be stored at 20 C upon arrival All other solutions and kit components should be kept tightly sealed and stored at room temperature All solutions and plastics can be used until the expiration date specified on their labels Precautions 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 The Binding Solution 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 Total RNA Purification Kit Dx Benchtop microcentrifuge 96 100 ethanol Xylene histological grade B mercaptoethanol optional 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 105 r where RCF required gravitational acceleration relative centrifugal force in units of g r radi
12. up to 600 uL of the lysate with the ethanol from Step 2 onto the column and centrifuge for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube Repeat Step 3b and 3c until all lysate has passed through the column Optional Step Norgen s FFPE RNA Purification 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 Column Wash a 2Q205 h Apply 400 uL of Wash Solution to the column and centrifuge for 1 minute 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 Discard the flowthrough and reassemble the spin column with its collection tube Apply 400 uL of Wash Solution to the column and centrifuge for 1 minute Discard the flowthrough and reassemble the spin column with its collection tube Wash column a third time by adding another 400 uL of Wash Solution and centrifuging for 1 minute Discard the flowthrough and reassemble the spin column with its collection tube Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 5 RNA Elution a b C
13. us of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force 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 The RNA area should be located away from microbiological work stations 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 There should be designated solutions tips tubes lab coats pipettes etc for RNA only All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water Clean all surfaces with commercially available RNase decontamination solutions When working with purified RNA samples ensure that they remain on ice during downstream applications Flow Chart Procedure for Purifying Total RNA using Norgen s FFPE RNA Purification Kit Dx FFPE Tissue Samples Deparaffinization with xylene Wash with ethanol Add Digestion Buffer Proteinase K Incubate Add Binding Solution Ethanol _ aes SS M Bind RNA in SPIN 3 Ii Wash RNA i SPIN 3 Elute RNA
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