21200 - Protocol (50 prep)
Contents
1. gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com 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 to 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 as proteins without the use of phenol or chloroform For le2. A that may affect sensitive downstream applications It is recommended Norgen s RNase Free DNase Kit Product 25710 be used for this step 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 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 Leukocyte RNA Isolation Procedure up to and including Binding to Column Step 3 3 Apply 400 uL of Wash Solution A to the column and centrifuge for 2 minutes 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 c
3. Lysis Buffer Vortex 1 Centrifuge to pellet cells 2 Gently decant supernatant a e White Leukocyte Pellet Flow Chart 2 Procedure for Total Leukocyte RNA Purification Lyse leukocyte pellet using Buffer RL Add Ethanol A Bind RNA to column Wash RNA three times with Wash Solution A AAL ach AAL aM Elute RNA with ni Elution Solution A q 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 Notes Prior to Use 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 t
4. and Product Stability All solutions should be kept tightly sealed and stored at room temperature The RBC Lysis Buffer should be stored at 4 C upon arrival These reagents should remain stable for at least 1 year in their unopened containers Advantages e Fast and easy processing using rapid spin column format No phenol 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 Component Product 21200 50 preps RBC Lysis Buffer 2x 100 mL Buffer RL 30 mL Wash Solution A 38 mL Elution Solution A 6 mL Mini Spin Columns 50 Collection Tubes 50 Elution tubes 1 7 mL 50 Product Insert 1 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 Suppl
5. ed during the use of the kit Ensure RNase proper procedures are followed when working with RNA contamination Please refer to Working with RNA at the beginning of this user guide Procedure nol In order to maintain the integrity of the RNA it is important performed that the procedure be performed quickly quickly enough RNA is Degraded Improper For short term storage RNA samples may be stored at storage of the 20 C for a few days It is recommended that samples be purified RNA stored at 70 C for longer term storage Leukocyte pellets generated at the end of Step 1 may be Leukocyte stored for up to 2 weeks at 70 C and used in this pellets were too old procedure Itis not recommended that samples be frozen for longer than 2 weeks as the integrity of the RNA may be compromised RNA was not washed 3 times with the 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 ANE H applications and thus must be washed from the column downstream applications Ensure that the dry spin under the Column Wash procedure Ethanol is performed in order to remove traces of ethanol prior to carryover elution Ethanol is known to interfere with many downstream applications Residual Large amounts Perform RNase free DNasel digestion on the RNA sample genomic DNA of genomic DNA after elution to remove genomic DNA contamina
6. hat all solutions 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 Prepare an appropriate amount of Buffer RL by adding 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 e It is recommended that no more than 2 mL of blood be used in order to prevent possible clogging of the column e 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 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 Frozen leukoc
7. ied Reagents and Equipment You must have the following in order to use the Leukocyte RNA Purification Kit e Benchtop microcentrifuge e mercaptoethanol e 96 100 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 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 3 1 Centrifuge to pellet cells 2 Gently decant supernatant Add 2 Volumes of RBC
8. olumn assembly at 25 30 C for 15 minutes 7 Without further centrifugation proceed directly to the second wash in the Column Wash section Step 4c Troubleshooting Guide Problem Possible Cause Solution and Explanation Incomplete lysis Ensure that the appropriate amount of Buffer RL was used of leukocytes to lyse the leukocyte pellet Ensure that the blood sample is collected with the iveieatred appropriate amount of EDTA which will prevent coagulation TON calls was of the red blood cells and allow for proper lysis Also check incomnl te that the appropriate amount of RBC Lysis Buffer is added to B the blood sample and that it is mixed and incubated properly Ethanol was not 4 added to the Ensure that 200 pL of 96 100 ethanol is added to the Poor RNA lysate lysate before binding to the column Recovery SES no Ensure that 90 mL of 96 100 ethanol is added to the Wash Solution A supplied Wash Solution A prior to use A ae It is recommended that the Elution Solution A supplied with Was uae this kit be used for maximum RNA recovery Do not exceed 2 mL of blood or 3 x 10 leukocytes per The column has column The amount of blood used may need to be become clogged decreased if the column shows clogging below the recommended level See also Clogged Column below Incomplete lysis Ensure that the appropriate amount of Buffer RL was used of leukocytes to lyse the leukocyte pellet Maeei
9. ot passed spin for an additional minute b Discard the flowthrough and reassemble the spin column with its collection tube c Repeat steps 4a and 4b 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 e 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 Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 50 uL of Elution Solution A to the column c 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 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 Appendix A Protocol for Optional On Column DNA Removal Norgen s Leukocyte RNA Purification Kit isolates leukocyte RNA with minimal amounts of genomic DNA contamination However an optional protocol is provided below for maximum rem that li oval of residual DN
10. rtexing for 10 seconds Note For input amounts greater than 500 uL of blood or 10 leukocytes it is recommended that the lysate is passed through a 25 gauge needle attached to a syringe 5 10 times at this point in order to shear the genomic DNA prior to loading onto the column 3 Binding to Column a Assemble a column with one of the provided collection tubes b Apply the lysate with the ethanol 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 Optional Step Norgen s Leukocyte RNA Purification Kit isolates total leukocyte 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 It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step 4 Column Wash a Apply 400 uL of Wash Solution A to the column and centrifuge for 1 minute Note Ensure the entire Wash Solution A has passed through into the collection tube by inspecting the column If the entire wash volume has n
11. ted Ensure that the blood sample is collected with the heer cells wae appropriate amount of EDTA which will prevent coagulation incomplete of the red blood cells and allow for proper lysis Improperly R lysed red blood cells will clog the column Clogged Column Amount of blood used exceeds kit specifications It is recommended that no more than 2 mL of blood or 3 x 10 leukocytes be used in order to prevent possible clogging of the column 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 St ees The solution should become a translucent red colour after Not Become Incomplete red RBC Lysis Solution has been added and incubated with the Clear Red blood cell lysis blood If not pellet the leukocytes and remove as much of During RBC the supernatant as possible Add another 5 volumes of Lysi RBC Lysis solution and incubate again ysis The leukocyte pellet should be white with only residual Leukocyte Incomplete red traces of red blood cells If red blood cell lysis is incomplete ellet is red blood celliyeis the pellet will be red In this case resuspend the leukocyte p y pellet in another 5 volumes of RBC Lysis Solution and incubate at room temperature for another 5 minutes RNases may be introduc
12. tion It is zontaminalioh in starting recommended that Norgen s RNase Free DNase Kit material Product 25710 be used for this step Related Products Product RNase Free DNase Kit 25710 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 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 P121200 20 M14 10
13. ukocyte 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 pages 4 and 5 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 will be removed in the flowthrough or retained on the top of the resin The bound RNA is then washed three times with the provided Wash Solution A in order to remove any remaining impurities and the purified leukocyte RNA is eluted with the Elution Solution A 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 RNase protection and primer extension and expression array assays Specifications Kit Specifications Maximum Column Binding Capacity 50 ug Maximum Column Loading Volume 650 uL Size of RNA Purified 2r ineluding SMARA Maximum Blood Input 2 mL or 3 x 10 Leukocytes Minimum Blood Input 10 uL Time to Complete 10 Purifications 40 minutes Average Yield 500 uL human blood 1 5 ug Storage Conditions
14. yte pellets should not be thawed prior to beginning the protocol Add the Binding Buffer directly to the frozen pellet Step 2a e It is important to work quickly during this procedure 1 Red Blood Cell Lysis a Add 5 volumes of RBC Lysis Buffer to blood samples collected with EDTA i e Add 2 5 mL of RBC Lysis Buffer to 500 uL of blood b 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 c Centrifuge at 250 x g 2 000 RPM for 3 minutes and decant supernatant d Add 2 additional volumes of RBC Lysis Buffer to pelleted white blood cells and mix by gentle vortexing for 10 seconds i e Add 1 mL of RBC Lysis Buffer to every 500 uL of input blood volume e 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 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 at the back of the manual if this occurs 2 Cell Lysate Preparation a Add 350 uL of Buffer RL directly to pelleted leukocytes b Lyse cells by gentle vortexing until homogeneity is reached c Add 200 uL of 96 100 ethanol provided by the user to the mixture and mix by vo
Download Pdf Manuals
Related Search