User Manual
Contents
1. The leukocyte pellet should be white If the pellet is red then the red blood cell lysis procedure was incomplete Please refer to the troubleshooting guide if this occurs 2 Cell Lysate Preparation of Add 350 uL of Binding Solution directly to pelleted leukocytes Lyse cells by gentle vortexing until homogeneity is reached Add 200 uL of 95 100 ethanol provided by the user to the mixture and mix by vortexing for 10 seconds Spin lysate for 2 minutes in a benchtop microcentrifuge to pellet any cell debris 3 Binding to Column a b Apply the clarified lysate from step 2d onto the column and centrifuge for 1 minute Discard the flowthrough Reassemble the spin column with its collection tube 4 Column Wash a 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 b C d Wash column a second time by adding another 400 uL of Wash Solution and centrifuging for 2 minutes Ensure that the column is dry Spin for an additional minute if necessary Discard the collection tube with the flowthrough 5 RNA Elution Place the column into a fresh 1 7 mL Elution tube provided with the kit a b Add 50 uL of Elution Buffer to the column c Centrifuge for 3 minutes at 5 200 x g 8 000 RPM Note the volu2. For leukocyte RNA purification whole blood samples are first collected with anticoagulants The red blood cells are removed through differential red blood cell lysis and the leukocytes are recovered by centrifugation please see flow charts on following pages The recovered leukcoytes are then lysed and the lysate is loaded onto a supplied 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 and genomic DNA will be removed in the flowthrough or retained on the top of the resin The bound RNA is then washed twice with the provided wash buffer in order to remove any remaining impurities and the purified leukocyte RNA is eluted with the elution buffer Norgen s kit allows for the isolation of total leukocyte RNA including all small RNA species The purified RNA is of the highest quality and can be used in a number of downstream applications including real time PCR reverse transcription PCR northern blotting RNaes protection and primer extension and expression array assays Specifications Kit Specifications Column Binding Capacity 50 ug Maximum Column Loading Volume 700 uL All sizes including small RNA Size of RNA Purified lt 200 nt Maximum Blood Input 500 uL Time to Complete 10 purifications Average Yield 500 uL human blood 1 5 ug Advantages e Fast and easy processing using rapid spin column format No phenol
3. The amount of The column has blood used may need to be decreased if the column shows become clogged clogging below the recommended level See also Clogged Column below Incomplete lysis Ensure that the appropriate amount of Binding Solution was of leukocytes used to lyse the leukocyte pellet Ensure that the blood sample is collected with the appropriate amount of EDTA which will prevent coagulation of the red blood cells and allow for proper lysis Improperly lysed red blood cells will clog the column Lysis of red blood cells was incomplete Clogged Amount of blood Column used exceeds kit specifications It is recommended that no more than 500 uL of blood be used in order to prevent possible clogging of the column Clarified lysate Ensure that the sample is centrifuged for at least 2 minutes was not used for after lysis and that only the clarified lysate is applied to the the binding step column Ensure that the centrifuge remains at room temperature throughout the procedure Temperatures below 20 C may Cause precipitates to form that can cause the columns to clog Centrifuge temperature too low Possible Cause Solution and Explanation Cloudy Pink Solution Does Not Become Clear Red During RBC Lysis Leukocyte pellet is red RNA is Degraded RNA does not perform well in downstream applications Residual genomic DNA contamination Incomplete red blood cell lysis Incomplete red bl
4. Fax 905 227 1061 BIOTEK wi CORPORATION Email techsupport norgenbiotek com gt 344 Merritt Street R F N St Catharines ON Canada L2T 1K6 gt Phone 866 667 4362 e 905 227 8848 Leukocyte RNA Purification Kit Product Insert Product 21200 Norgen s Leukocyte RNA Purification Kit provides a rapid method for the isolation and purification of total leukocyte white blood cell RNA from mammalian blood samples RNA isolated from blood can be used in various expression studies including those focusing on diseases However a major problem with blood RNA isolation is that a large portion of the RNA present is globin mRNA which is found primarily in red blood cells In fact up to 70 of the mass of mRNA in whole blood total RNA is globin transcripts Therefore it is desirable to be able to remove the red blood cells from the sample and isolate only the RNA associated with the leukocytes which will result in improved expression profiling and other applications by removing the masking effects of this abundant globin mRNA Norgen s Leukocyte RNA Purification Kit can be used isolate and purify total leukocyte RNA including all small RNAs from mammalian blood samples 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 the other cellular components such proteins without the use of phenol or chloroform
5. claving 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 Flow Chart 1 Procedure for Differential Red Blood Cell RBC Lysis Collect Blood in 4 8mM EDTA Add 5 Volumes of RBC Lysis Buffer Vortex and incubate for 3 5 minutes 1 Centrifuge to pellet cells 2 Gently decant supernatant Add 2 Volumes of RBC Lysis Buffer Vortex 1 Centrifuge to pellet cells 2 Gently decant supernatant White Leukocyte Pellet Flow Chart 2 Procedure for Total Leukocyte RNA Purification Lyse leukocyte pellet using Binding Solution Add Ethanol Centrifuge to clear lysa
6. me eluted from the column If the entire 50 uL 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 a 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 Troubleshooting Guide Possible Cause Solution and Explanation Incomplete lysis Ensure that the appropriate amount of Binding Solution was of leukocytes used to lyse the leukocyte pellet Ensure that the blood sample is collected with the appropriate amount of EDTA which will prevent coagulation of the red blood cells and allow for proper lysis Also check that the appropriate amount of RBC Lysis Buffer is added to the blood sample and that it is mixed and incubated properly Lysis of red blood cells was incomplete Ethanol was not added to the lysate Ensure that 200 uL of 95 100 ethanol is added to the Poor RNA lysate before binding to the column Recovery Ethanol was not added to the Wash Solution Ensure that 35 mL of 95 ethanol is added to the supplied Wash Solution prior to use An alternative elution solution was used It is recommended that the Elution Buffer supplied with this kit be used for maximum RNA recovery Do not exceed 500 uL of blood per column
7. mples be frozen for longer than 2 weeks as the integrity of the RNA will be compromised Traces of salt from the binding step may remain in the sample if the column is not washed twice 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 Perform DNasel digestion on the RNA sample after elution to remove genomic DNA contamination Related Products Product Total RNA Purification Kit 17200 Cytoplasmic amp Nuclear RNA Purification Kit 21000 microRNA Purification Kit 21300 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 344 Merritt St St Catharines ON Canada L2T 1K6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2006 Norgen Biotek Corp P1l21000 5
8. ood cell lysis RNase contamination Procedure not performed quickly enough Improper storage of the purified RNA Leukocyte pellets were too old RNA was not washed twice with the provided Wash Solution Ethanol carryover Large amounts of genomic DNA in starting material The solution should become a translucent red colour after RBC Lysis Solution has been added and incubated with the blood If not pellet the leukocytes and remove as much of the supernatant as possible Add another 5 volumes of RBC Lysis solution and incubate again The leukocyte pellet should be white with only residual traces of red blood cells If red blood cell lysis is incomplete the pellet will be red In this case resuspend the leukocyte pellet in another 5 volumes of RBC Lysis Solution and incubate at room temperature for another 5 minutes 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 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 Leukocyte pellets generated at the end of Step 1 may be stored for up to 2 weeks at 70 C and used in this procedure It is not recommended that sa
9. or chloroform extractions Differential red blood cell lysis allows for the removal of a majority of globin mRNAs Isolate total leukocyte RNA including all small RNA species High quality leukocyte RNA can be used in a number of downstream applications Kit Components Micro Spin Columns assembled with 2 mL collection 50 tubes Elution tubes 1 7 ml ee 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 User must determine the suitability of the product for their particular use This kit is intended for research purposes only and not for human or drug use This kit is not designed for diagnostic purposes MSDS sheets are available upon request 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 Ensure that a proper lab coat disposable gloves and protective eyewear are worn when working with this kit Customer Supplied Reagents and Equipment You must have the following in order to use the Leukocyte RNA Purification Kit e Benchtop microcentrifuge e mercaptoethanol e 95 100 ethanol Working with RNA RNases are very stable and robust enzymes that degrade RNA Auto
10. te Bind RNA Wash RNA Elute RNA SPIN 3 Total Leukocyte RNA Procedure 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 Protocol for Total RNA Purification from Isolated Leukocytes 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 Notes Prior to Use e Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of the Wash Solution by adding 35 mL of 95 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 50 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Prepare an appropriate amount of Binding Buffer by adding 10 uL of B mercaptoethanol provided by the user to each 1 mL of Binding Buffer required mercap
11. toethanol is toxic and should be dispensed in a fume hood e It is recommended that no more than 500 uL of blood be used in order to prevent possible clogging of the column e Blood samples should be collected into a tube containing EDTA such that the final concentration of the EDTA is 4 8 mM e Only fresh blood can be used with this procedure Frozen whole blood can not be used e For optimal results blood samples should be processed within a few hours of collection e Leukocyte pellets generated in the first step can be used directly in the procedure or stored at 70 C for later use Pellets should be stored for no longer than 2 weeks to ensure that the integrity of the RNA is not compromised e It is important to work quickly during this procedure 1 Red Blood Cell Lysis ap Add 5 volumes of RBC Lysis Buffer to blood samples collected with EDTA Incubate at room temperature for 3 to 5 minutes with brief vortexing during the incubation to mix Note Ensure that the solution changes from a milky opaque pink to clear red before proceeding to the next step Centrifuge at 250 x g 2 000 RPM for 3 minutes and decant supernatant Add 2 additional volumes of RBC Lysis Buffer to pelleted white blood cells and mix by gentle vortexing for 10 seconds Centrifuge at 250 x g 2 000 RPM for 3 minutes and decant supernatant A few uL of media may be left behind with the pellet in order to ensure that the pellet is not dislodged Note
Download Pdf Manuals
Related Search