Protocol - Norgen Biotek Corp.
Contents
1. d Depending on your lysate volume repeat Step 3b and 3c as necessary 4 Column Wash a Apply 400 uL of Wash Solution A to the column and centrifuge at 14 000 x g 14 000 RPM 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 not 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 Repeat steps 4a and 4b to wash column a third time e 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 40 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 20 uL is recommended c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by 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 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 sample may be stored at 20 C for a few days It is recommended that samples be placed a2. please see the flow chart on page 4 The DNA is then captured on a Genomic DNA Removal Column Ethanol is then added to the flowthrough of the DNA removal step and the solution is loaded onto an RNA Purification Micro 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 20 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 35 ug Maximum Column Loading Volume 650 uL Minimum Elution Volume 20 uL Size of RNA Purified All sizes including small RNA lt 200 nt Maximum Amount of Starting Material Animal Cells 5 x 10 cells Animal Tissues 3 mg for most tissues Laser Captured Microdissection LCM Up to 5 x 10 cells Time to Complete 10 Purifications 22 minutes Average Yields HeLa Cells 1 x 10 cells 1 5 ug for fibrous tissue an additional Proteinase K treatment is required Advantages Fast and easy processing using rapid micro spin col
3. uL of media may be left behind in order to ensure that the pellet which could be invisible is not dislodged 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 Proceed to Step 2 1B Lysate Preparation from Animal Tissues Notes Prior to Use Norgen s Total RNA Purification Plus Micro Kit is designed for isolating RNA from small amounts of non fibrous tissue samples up to 3 mg in most cases If a larger amount of starting material or fibrous tissue is desired an additional Proteinase K treatment is required Please refer to Appendix A for instruction RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized Thus it is important that the procedure is carried out as quickly as possible particularly the Cell Lysate Preparation step Fresh or frozen tissues may be used for the procedure Tissues should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Tissues may be stored at 70 C for several months When isolating total RNA from frozen tissues ensure that the tissue does not thaw during weighing or prior to grinding with the mortar and pestle Tissues stored in RNA stabilization reagents such as RNAlater are compatible with this isolation procedure Prior to isolation carefully remove the tissue from the storage reagent using f
4. A samples ensure that they remain on ice during downstream applications Flowchart Procedure for Purifying Total RNA using Norgen s Total RNA Purification Plus Micro Kit A Genomic DNA Removal Lyse cells or tissue using Buffer RL f Eliminate DNA using the Genomic DNA Removal Column SPIN 3 B Purification of RNA r 6 Add ethanol Bind to RNA Purification Micro Column Flowthrough with RNA SPIN D A Wash three times with Wash Solution A SPIN A AL Elute RNA with Elution Solution A SPIN J 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 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 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 pr
5. Ty R 3430 Schmon Parkway S N E N Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com Total RNA Purification Plus Micro Kit Product Insert Product 48500 Norgen s Total RNA Purification Plus Micro Kit provides a rapid and sensitive method for the isolation and purification of total RNA from small input amounts of cultured animal cells tissue samples and microdissected samples including laser capture microdissection LCM The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA Genomic DNA is removed from the sample using a Genomic DNA Removal column and 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 or tissue of interest with the provided Buffer RL
6. dd 600 uL of RNase Free Water not provided to the lysate Vortex to mix Add 20 uL of reconstituted Proteinase K to the lysate and incubate at 55 C for 15 minutes Vortex the tubes occasionally during incubation Spin the lysate for 2 minutes to pellet any cell debris Transfer the supernatant to another RNase free microcentrifuge tube Note the volume Proceed to Step 2 Troubleshooting Guide 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 Column Aas materials The amount of starting material may need to Become clodaed be decreased if the column shows clogging below the 99 recommended levels See also Clogged Column below ee ega was It is recommended that the Elution Solution A supplied used with this kit be used for maximum RNA recovery Ethanolwas ot Ensure that the appropriate amount of ethanol is added to the lysate before binding to the RNA Purification Micro added to the lysate column Poor RNA RETA cane Ensure that 90 mL of 96 100 ethanol is added to the Recovery supplied Wash Solution A prior to use Solution A Low RNA content in cells or tissues used Different tissues and cells have different RNA contents and thus the expected yield of RNA will vary greatly from these different sources Please check literatu
7. en in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Tissues may be stored at 70 C for several months When isolating total RNA from frozen tissues ensure that the tissue does not thaw during weighing or prior to grinding with the mortar and pestle e Tissues stored in RNA stabilization reagents such as RNAlater are compatible with this isolation procedure Prior to isolation carefully remove the tissue from the storage reagent using forceps and dry any excessive liquid e This protocol is particularly suitable for isolating RNA from up to 7 5 mg of tissues including fibrous connective tissues Cell Lysate Preparation a Excise the tissue sample from the animal b Determine the amount of tissue by weighing c Transfer the tissue into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the tissue thoroughly using a pestle Note The use of liquid nitrogen is recommended However if homogenization without flash freezing is preferred proceed to Step e d Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 300 uL of Buffer RL to the tissue sample and continue to grind until the sample has been homogenized Note Maximum homogenization may be achieved by passing the lysate 5 10 times through a 25 gauge needle attached to a syringe Using a pipette transfer the lysate into an RNase free microcentrifuge tube not provided A
8. eparing 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 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 Optional The use of B mercaptoethanol in lysis is highly recommended for most animal tissues particularly those known to have a high RNAse content ex pancreas including LCM samples 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 Cultured Animal Cells Notes Prior to Use e The maximu
9. m recommended input of cells is 5 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 12 well plate of HeLa cells will contain 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 1A ii c 1A i Cell Lysate Preparation from Cells Growing in a Monolayer a b Aspirate media and wash cell monolayer with an appropriate amount of PBS Aspirate PBS Add 350 uL of Buffer RL directly to culture plate Lyse cells by gently tapping culture dish and swirling buffer around plate surface for five minutes Transfer lysate to a microcentrifuge tube Proceed to Step 2 Cell Lysate Preparation from Cells Growing in Suspension and Lifted Cells 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 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
10. must be washed from the column in downstream Ensure that the dry spin under the Column Wash applications procedure is performed in order to remove traces of Ethanol carryover ethanol prior to elution Ethanol is known to interfere with many downstream applications 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 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 P148500 2 M14 12
11. orceps and dry excessive liquid The maximum recommended input of tissue varies depending on the type of tissue being used Please refer to Table 1 below as a guideline for maximum tissue input amounts If your tissue of interest is not included in the table below we recommend starting with an input of no more than 3 mg Table 1 Recommended Maximum Input Amounts of Different Tissues Tissue Maximum Input Amount Brain Kidney Liver Lung 3 mg Spleen Heart Muscle Refer to Appendix A 1B Cell Lysate Preparation from Animal Tissues a Excise the tissue sample from the animal b Determine the amount of tissue by weighing Please refer to Table 1 for the recommended maximum input amounts of different tissues For tissues not included in the table we recommend starting with an input of no more than 3 mg c Transfer the tissue into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the tissue thoroughly using a pestle d Allow the liquid nitrogen to evaporate without allowing the tissue to thaw e Add 400 uL of Buffer RL to the tissue sample and continue to grind until the sample has been homogenized Homogenize by passing the lysate 5 10 times through a 25 gauge needle attached to a syringe f Using a pipette transfer the lysate into an RNase free microcentrifuge tube not provided g Spin the lysate for 2 minutes to pellet any cell debris Transfer the supernatant to anothe
12. predominately white contents o RNA Purification Micro Columns collection tubes provided separately column has predominately black contents 2 Genomic DNA Removal a Retrieve a gDNA Removal Column pre assembled with a collection tube b Apply up to 600 uL of the lysate prepared from Section 1 onto the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute 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 c Retain the flowthrough for RNA Purification Section 3 The flowthough contains the RNA and should be stored on ice or at 20 C until the RNA Purification protocol is carried out d Dispose of the gDNA Removal Column with the bound gDNA Section 3 Total RNA Purification from All Types of Lysate 3 Binding RNA to Column a To every 100 uL of flowthrough from Step 2c add 60 uL of 96 100 Ethanol Mix by vortexing Note For example for 300 uL of flowthrough add 180 uL of 96 100 Ethanol b Assemble an RNA Purification Micro Column with one of the provided collection tubes c Apply up to 600 uL of the lysate with the ethanol onto the column and centrifuge at 14 000 x g 14 000 RPM for 1 minute 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
13. r RNase free microcentrifuge tube Note the volume of the supernatant lysate h 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 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 Atthe 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 e Proceed to Step 2 Section 2 Genomic DNA Removal from All Types of Lysate Notes e 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 e This kit is provided with 2 separate columns When columns are removed from the labeled bags they are supplied in they can easily be identified as follows o gDNA Removal Columns provided with collection tubes attached column has
14. re to determine the expected RNA content of your starting material Cell Culture Cell monolayer was not washed with PBS Ensure that the cell monolayer is washed with the appropriate amount of PBS in order to remove residual media from cells LCM Sample was not incubated at 42 C for 30 minutes Ensure that the incubation at 42 C for the removal and lysis of cells from the thermoplastic film 10 Problem Possible Cause Solution and Explanation Insufficient solubilization of cells or tissues Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue Perform the optional Proteinase K treatment for fibrous tissues Maximum number of cells or amount of tissue exceeds kit specifications Refer to specifications to determine if amount of starting material falls within kit specifications Clogged Column The lysate may be passed through a 25 gauge needle High amounts of hed 5 10 ti nord h h enomic DNA attac e to a syringe 5 0 times in order to s ear t e 9 genomic DNA prior to loading onto the Genomic DNA present in sample Removal column Cent f a Ensure that the centrifuge remains at room temperature g throughout the procedure Temperatures below 15 C temperature too a bw may cause precipitates to form that can cause the columns to clog RNases may be introduced during the use of the kit RNase Ensure proper procedures are follo
15. t 70 C for long term storage Appendix A Lysate Preparation from Animal Tissues with the use of Proteinase K Customer Supplied Reagent e RNase Free Proteinase K e RNase Free Water e f mercaptoethanol Notes Prior to Use e Ensure that all solutions are at room temperature prior to use e Werecommend the use of Norgen s Proteinase K Cat 17904 for this step Reconstitute each of the Proteinase K vials in 600 uL of molecular biology grade water or 10 mM Tris HCl pH 7 5 RNase Free For every isolation 20 uL of the reconstituted Proteinase K is needed Aliquot the remainder into small fractions and store the unused portions at 20 C until needed e f using another source of Proteinase K reconstitute in molecular biology grade water or 10 mM Tris HCl pH 7 5 RNase Free to give a 20 mg mL final concentration For every isolation 20 uL of the reconstituted Proteinase K is needed Aliquot the remainder into small fractions and store the unused portions at 20 C until needed e 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 e RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized Thus it is important that the procedure is carried out as quickly as possible particularly the Cell Lysate Preparation step e Fresh or frozen tissues may be used for the procedure Tissues should be flash froz
16. umn format Small elution volume of 20 uL Isolate total RNA from large rRNA down to microRNA miRNA without compromising total yield No phenol or chloroform extractions Isolate high quality total RNA from a variety of sources Rapidly remove contaminating genomic DNA without the use of enzymes RNA can be isolated and detected from as little as a single animal cell Kit Components Component Product 48500 50 preps Buffer RL 40 mL Wash Solution A 38 mL Elution Solution A 6 mL Genomic DNA Removal Column 50 RNA Purification Micro Column 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 guanidinium salts and should be handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properl
17. wed 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 i ially i for the Cell L P l erformed quickly is especially important or the Ce ysate reparation p Step in the Animal Tissue protocol since the RNA in enough NA animal tissues is not protected after harvesting until it is disrupted and homogenized RNA is Improper storage of For short term storage RNA samples may be stored at Degraded the purified RNA 20 C for a few days Itis recommended that samples be stored at 70 C for longer term storage Frozen tissues or cell pellets were allowed to thaw prior to RNA isolation Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised Starting material may have a high RNase content For starting materials with high RNAase content it is recommended that B mercaptoethanol be added to the Buffer RL 11 Problem Possible Cause Solution and Explanation RNA was not washed 3 times RNA does not with the provided 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 perform well Wash Solution A applications and thus
18. y dispose of any of these solutions Customer Supplied Reagents and Equipment You must have the following in order to use the Total RNA Purification Plus Micro Kit For All Protocols e Benchtop microcentrifuge e 96 100 ethanol e mercaptoethanol optional For Animal Cell Protocol e PBS RNase free For Animal Tissue Protocol e Liquid nitrogen e Mortar and pestle e 70 ethanol For Laser Captured Microdissection LCM Protocol e Sterile fine forceps e Water bath or heat block set at 42 C 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 RN
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