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1. Problem Possible Cause Solution and Explanation Incomplete lysis of Ensure that the appropriate amount of Buffer RL was cells or tissue used for the amount of cells or tissue Do not exceed the recommended amounts of starting Gol n has materials The amount of starting material may need to become clogged be decreased if the column shows clogging below the 99 recommended levels See also Clogged Column below Pe Was It is recommended that the Elution Solution A supplied need with this kit be used for maximum RNA recovery Ethanol was not Ensure that the appropriate amount of ethanol is added added to the lysate to the lysate before binding to the column Poor RNA Ethanol Recovery Tanp Wae nor Ensure that 90 mL of 96 100 ethanol is added to the added to the Wash supplied Wash Solution A prior to use Solution A Different tissues and cells have different RNA contents Low RNA content and thus the expected yield of RNA will vary greatly from in cells or tissues these different sources Please check literature to used determine the expected RNA content of your starting material Cell Culture Cell Ensure that the cell monolayer is washed with the monolayer was not appropriate amount of PBS in order to remove residual washed with PBS media from cells LCM Sample was not incubated at Ensure that the incubation at 42 C for the removal and 42 C for 30 lysis of cells from the thermoplastic film minutes Centrifuge E2. 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 1B ii Cell Lysate Preparation from Cells Growing in Suspension and Lifted Cells a Transfer cell suspension to an RNase free tube not provided and centrifuge at no more than 200 x g 2 000 RPM for 10 minutes to pellet cells b Carefully decant the supernatant Note For inputs of over 10 cells 5 10 uL of media may be left behind with the pellet in order to ensure that the pellet is not dislodged For inputs of fewer than 10 cells 30 50 uL of media may be left behind in order to ensure that the pellet which could be invisible is not dislodged c Add 350 uL of Buffer RL to the pellet Lyse cells by vortexing for 15 seconds Ensure that the entire pellet is completely dissolved before proceeding to the next step d Add 200 uL of 96 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 1C Lysate Preparation from Laser Captured Microdissection LCM Notes Prior to Use e LCM samples obtained from frozen sections are recommended Formalin Fixed Paraffin Embedded sections may also be used However RNA isolated from FFPE samples generally has poorer quality than that from frozen sections 1C Cell Lysate Preparation from Laser Captured Microdissection LCM a Aliquot 300 uL of Buffer RL to an RNase free microcentrifuge tube b Remove the thermoplastic film containing the
3. 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 Single Cell RNA Purification Kit Product Insert Product 51800 Norgen s Single Cell RNA Purification Kit provides a rapid and sensitive method for the isolation and purification of total RNA from small input amounts of cultured animal cells sorted cells and microdissected samples including laser capture microdissection LCM The kit can recover RNA from as little as a single cell to 2 x 10 cells The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA The RNA is preferentially purified from other cellular components such as proteins 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 Norgen s Purification Technology Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix The RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform The process involves first lysing the cells with the provided Buffer RL please see the flow chart on page 4 Ethano
4. A 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 It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step This step should be performed at this point in the protocol 3 Column Wash a Apply 400 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 A 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 Discard the flowthrough and reassemble the spin column with its collection tube Repeat steps 3a and 3b to wash column a second time d Wash column a third time by adding another 400 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 9 f Spin the column for 2 minutes at 14 000 x g 14 000 RPM in order to thoroughly dry the resin Discard the collection tube 4 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 8 20 uL of Elution Solution A to the column Note For higher concentrations of RNA a lower elution volume may be used A minimum volume of 8 uL is recommended c Centrifuge for 1 minutes at 200 x g 2 000 RPM followed by 1
5. captured cells using sterile fine forceps Carefully submerge the sample into the aliquoted Buffer RL Close the microcentrifuge cap c Incubate the sample at 42 C for 30 minutes Apply vortex for 15 seconds after every 10 minutes d At the end of the incubation vortex the tube one more time for 15 seconds The thermoplastic film may be removed at this point using sterile fine forceps Otherwise proceed to Step 1Ce e Add 300 uL of 70 ethanol provided by the user to the lysate Vortex to mix Proceed to Step 2 Section 2 Total RNA Purification from All Types of Lysate Note The remaining steps of the procedure for the purification of total RNA are the same from this point forward for all the different types of lysate 2 Binding RNA to Column a Assemble a Single Cell RNA Spin Column with one of the provided collection tubes b Apply up to 600 uL of the lysate with the ethanol from Step 1 onto the column and centrifuge for 1 minute at 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 Depending on your lysate volume repeat Step 2b and 2c as necessary Optional Step Norgen s Single Cell RNA Purification Kit isolates total RNA with minimal amounts of genomic DN
6. chnical 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 2014 Norgen Biotek Corp P151800 3 M14 11
7. e 96 well collection plate for cell collection For Animal Cell Protocol e PBS RNase free For Laser Captured Microdissection LCM Protocol e Sterile fine forceps e Water bath or heat block set at 42 C e 70 ethanol 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 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 Flowchart Procedure for Purifying Total RNA using Norgen s Single Cell RNA Purification Kit Lyse cells or tissue using Buffer RL Add Ethanol Bind to column SPIN A AL ai lt Wash three times
8. ge tube or into each well of a 96 well collection plate b Sort the cells directly into the aliquoted Buffer RL Mix by pipetting up and down a few times If the cells are collected onto a 96 well plate transfer the lysate to an RNase free microcentrifuge tube c Add 100 uL of 70 ethanol provided by the user to the lysate Mix by vortexing 1B Lysate Preparation from Cultured Animal Cells Notes Prior to Use e The maximum recommended input of cells is 2 x 10 A hemocytometer can be used in conjunction with a microscope to count the number of cells As a general guideline each well of a confluent 96 well plate of HeLa cells will contain 2 5 x 10 cells e Cell pellets can be stored at 70 C for later use or used directly in the procedure Determine the number of cells present before freezing e Frozen pellets should be stored for no longer than 2 weeks to ensure that the integrity of the RNA is not compromised e Frozen cell pellets should not be thawed prior to beginning the protocol Add the Buffer RL directly to the frozen cell pellet Step 1B ii c 1B i Cell Lysate Preparation from Cells Growing in a Monolayer a Aspirate media and wash cell monolayer with an appropriate amount of PBS Aspirate PBS b Add 200 uL of Buffer RL directly to culture plate c Lyse cells by gently tapping culture dish and swirling buffer around plate surface for five minutes d Transfer lysate to a microcentrifuge tube e Add 120 uL of 96
9. hort term storage RNA samples may be stored at Degraded E ed BAA 20 C for a few days Itis recommended that samples P be stored at 70 C for longer term storage Frozen tissues or cell pellets were Do not allow frozen tissues to thaw prior to grinding with allowed to thaw the mortar and pestle in order to ensure that the integrity prior to RNA of the RNA is not compromised isolation Starting material For starting materials with high RNAase content it is may have a high recommended that B mercaptoethanol be added to the RNase content Buffer RL Genomic L r g amounts of Perform RNAse free DNasel digestion on the RNA DNA no mate al sample after elution to remove genomic DNA contamination used contamination It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step 10 Related Products Product Proteinase K 2 Vials 17904 RNase Free DNase Kit 25710 Total RNA Purification Kit 17200 Total RNA Purification 96 Well Kit 24300 Total RNA Purification Maxi Kit 26800 Animal Tissue RNA Purification Kit 25700 Plant Fungi Total RNA Purification Kit 25800 RNA Protein Purification Kit 24100 RNA DNA Protein Purification Kit 24000 Cytoplasmic amp Nuclear RNA Purification Kit 21000 Leukocyte RNA Purification Kit 21200 microRNA Purification Kit 21300 100b RNA Ladder 15002 1kb RNA Ladder 15003 Technical Support Contact our Te
10. l is then added to the lysate and the solution is loaded onto a Single Cell RNA Spin Column Norgen s resin binds RNA in a manner that depends on ionic concentrations Thus only the RNA will bind to the column while the contaminating proteins will be removed in the flowthrough or retained on the top of the resin The bound RNA is then washed with the provided Wash Solution A in order to remove any remaining impurities and the purified total RNA is eluted with the Elution Solution A The special design of the micro spin column allows a small elution volume of as little as 8 uL The purified RNA is of the highest integrity and can be used in a number of downstream applications Specifications Kit Specifications Maximum Column Binding Capacity 10 ug Maximum Column Loading Volume 650 uL Minimum Elution Volume 8 uL Size of RNA Purified All sizes including small RNA lt 200 nt Acceptable Amount of Starting Material Animal Cells Single Cell to 2 x 10 cells Laser Captured Microdissection LCM Up to 2 x 10 cells Time to Complete 10 Purifications 20 minutes Average Yields HeLa Cells 1 x 10 cells 1 5 ug HeLa Cells 100 cells 1 5 ng HeLa Cells 1 cell gt 1pg Advantages Fast and easy processing using rapid spin column format Small elution volume of 8 uL Isolate total RNA from large rRNA down to microRNA miRNA without compromising total yield No phenol or chloroform extractions Is
11. lutions are at room temperature prior to use e Prepare a working concentration of the Wash Solution A by adding 90 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution A This will give a final volume of 128 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Optional The use of B mercaptoethanol in lysis is highly recommended for most animal tissues LCM samples particularly those known to have a high RNAse content ex pancreas It is also recommended for users who wish to isolate RNA for sensitive downstream applications Add 10 uL of B mercaptoethanol provided by the user to each 1 mL of Buffer RL required B mercaptoethanol is toxic and should be dispensed in a fume hood Alternatively the Buffer RL can be used as provided e It is important to work quickly during this procedure 1A Lysate Preparation from Single Animal Cell Notes Prior to Use e Freshly sorted cells are recommended for this protocol e Cells can be sorted by various procedures including classic flow cytometry and fluorescence activated cell sorting FACS e Ensure that all the components of the flow cytometer such as dip tube septa flow cell all tubing lines and nozzles are decontaminated for RNases using an appropriate reagent 1A Cell Lysate Preparation from Single Cell or Sorted Cells a Aliquot 100 uL of Buffer RL to an RNase free microcentrifu
12. 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 5 Storage of RNA The purified RNA sample may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage 6 Quantification of RNA For cell inputs of 2 1 x 10 cells it is possible to quantify RNA using a nanospectrophotometer such as Thermo Scientific s NanoDrop For cell inputs between 10 and 10 cells RNA quantification requires a highly sensitive fluorescence based system such as Life Technologies RiboGreen RNA Assay kit For cell inputs between a single cell and 10 cells quantification could be performed using RT qPCR of an RNA transcript of high abundance such as GAPDH or 5S rRNA with a standard curve generated with total RNA of known concentration Appendix A Protocol for Optional On Column DNA Removal Norgen s Single Cell RNA Purification 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 f For every on column reaction to be performed prepare a mix of 15 uL of DNase I and 100 uL of Enzyme Incubation Buffer using Norgen
13. nsure that the centrifuge remains at room temperature Clogged g throughout the procedure Temperatures below 15 C temperature too oe Column may cause precipitates to form that can cause the low columns to clog Problem Possible Cause Solution and Explanation RNA does not RNA was not washed 3 times with the provided Wash Solution A Traces of salt from the binding step may remain in the sample if the column is not washed 3 times with Wash Solution A Salt may interfere with downstream applications and thus must be washed from the column perform well in stip cal Ensure that the dry spin under the Column Wash applications procedure is performed in order to remove traces of Emanolearyover ethanol prior to elution Ethanol is known to interfere with many downstream applications RNases may be introduced during the use of the kit RNase Ensure proper procedures are followed when working contamination 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 Procedure not is especially important for the Cell Lysate Preparation performed quickly i Step in the Animal Tissue protocol since the RNA in enough es animal tissues is not protected after harvesting until it is disrupted and homogenized RNA is impiober storage or For s
14. olate high quality total RNA from a variety of sources RNA can be isolated and detected from as little as a single animal cell Kit Components Component Product 51800 50 preps Buffer RL 40 mL Wash Solution A 38 mL Elution Solution A 6 mL Single Cell RNA Spin Columns 50 Collection Tubes 50 Elution tubes 1 7 mL 50 Product Insert 1 Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature 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 For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com with chemicals The Buffer RL 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 Single Cell RNA Purification Kit For All Protocols e Benchtop microcentrifuge e 96 100 ethanol e B mercaptoethanol optional For Single Cell Protocol
15. 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 uL RNase free DNase solution according to the manufacturer s instructions A 100 uL aliquot is required for each column to be treated 2 Perform the appropriate Total RNA Isolation Procedure for your starting material up to and including Binding to Column Steps 1 and 2 of all protocols 3 Apply 400 uL of Wash Solution A to the micro spin column and centrifuge for 2 minute Discard the flowthrough Reassemble the spin column with its collection tube 4 Apply 100 uL of the RNase free DNase solution prepared in Step 1 to the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute Note Ensure that the entire DNase solution passes through the column If needed spin at 14 000 x g 14 000 RPM for an additional minute 5 After the centrifugation in Step 4 pipette the flowthrough that is present in the collection tube back onto the top of the column Note Ensure Step 5 is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species 6 Incubate the column assembly at 25 30 C for 15 minutes 7 Without any further centrifugation proceed directly to the second wash step in the Column Wash section Step 3c Troubleshooting Guide
16. with Wash Solution A SPIN Elute RNA with Elution Solution A SPIN AL alm o Purified Total RNA 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 radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force Section 1 Preparation of Lysate From Various Cell Types Notes Prior to Use e The steps for preparing the lysate are different depending on the starting material Step 1 However the subsequent steps are the same in all cases Steps 2 6 e Please ensure that the correct procedure for preparing the lysate from your starting material is followed e 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 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 so
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