21300 - Protocol (25 prep)
Contents
1. High quality small RNA can be used in various downstream applications Kit Components Component Product 21300 25 preps Buffer RL 40 mL Wash Solution A 38 mL Elution Solution A 6 mL Large RNA Removal Column 25 microRNA Enrichment Column 25 Collection Tube 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 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 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 properly dispose of any of these solutions 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 Customer Supplied Reagents and Equipment For All Protocols e2. Removal a Assemble a Large RNA Removal Column with one of the provided collection tubes b Apply the lysate with the ethanol from step 1 onto the column and centrifuge for 1 minute at 14 000 x g 14 000 RPM Retain the flowthrough which contains the small RNA species If small RNA depleted RNA is to be isolated retain the column and proceed to the Optional Large RNA Purification Protocol Appendix A Otherwise discard the column c Transfer the flowthrough to an RNase free microcentrifuge tube not provided The flowthrough contains the small RNA thus ensure that this fraction is not discarded 3 Small RNA Capture a For animal cells bacteria or blood Add 350 uL of 96 100 ethanol provided by the user to the flowthrough collected in Step 2b Mix by vortexing for 10 seconds For animal tissues or plant Based on the lysate volume determined in Steps 1Bg or 1De add 1 volume of 96 100 ethanol provided by the user to the flowthrough collected in Step 2b 100 uL of ethanol is added to every 100 uL of flowthrough Mix by vortexing for 10 seconds b Assemble a microRNA Enrichment Column with one of the provided collection tubes c Apply half of the lysate mix with ethanol onto the column and centrifuge for 1 minute at 2 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
3. at 14 000 x g 14 000 RPM d Discard the flowthrough and reassemble the spin column with the collection tube e Repeat steps 3c and 3d to complete the capture of the small RNA 4 Column Wash a Apply 400 uL of Wash Solution A to the microRNA Enrichment 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 the collection tube 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 9 5 Small RNA Elution a Place the microRNA Enrichment Column into a fresh 1 7 mL Elution tube provided with the kit b Add 50 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 ad
4. centrifuging the lysate in order to pellet any debris and applying the clarified lysate to the Large RNA Removal Column Repeating the procedure should result in the removal of the large contaminating RNA species 13 Related Products Product 100b RNA Ladder 15002 1kb RNA Ladder 15003 Total RNA Purification Kit 17200 Cytoplasmic amp Nuclear RNA Purification Kit 21000 Leukocyte RNA Purification Kit 21200 RNA Protein Purification Kit 24100 RNA DNA Protein Purification Kit 24000 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 P121300 22 M14 14
5. 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 e The maximum recommended input of cells is 3 x 10 A hemocytometer can be used in conjunction with a microscope to count the number of cells As a general guideline a confluent 3 5 cm plate of HeLa cells will contain 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 pellets should not be thawed prior to beginning the protocol Add the Buffer RL directly to the frozen cell pellet Step A ii 1c 1A i Cell Lysate Preparation from Cells Growing in a Monolayer a b C d e Aspirate media and wash cell monolayer with an appropriate amount of PBS Aspirate PBS Add 300 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 RNase free microcentrifuge tube Add 150 uL of 96 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 1A ii Cell Lysate Preparation from Cells Growing in Suspension and Lifted Cells a b C d Transfer cell suspension to an RN
6. input of no more than 10 mg Table 1 Recommended Maximum Input Amounts of Different Tissues Tissue Maximum Input Amount Brain 25 mg Heart 5 mg Kidney 10 mg Liver 20 mg Lung 10 mg Spleen 10 mg 1B Cell Lysate Preparation from Animal Tissues a b Excise the tissue sample from the animal 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 10 mg 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 Allow the liquid nitrogen to evaporate without allowing the tissue to thaw 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 Using a pipette transfer the lysate into an RNase free microcentrifuge tube not provided Spin the lysate for 2 minutes to pellet any cell debris Transfer the supernatant to another RNase free microcentrifuge tube Note the volume of the supernatant lysate Add a volume of 96 100 ethanol provided by the user that is equivalent to 50 of the lysate volume 50 uL of ethanol is added to every 100 uL of lysate Vortex to mix Proceed to Step 2 1C Cell Lysate P
7. 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 2b and 2c 3 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 10 Troubleshooting Guide Problem Possible Cause Solution and Explanation Incomplete lysis of Ensure that the appropriate amount of Buffer RL was used cells or tissue for the amount of cells or tissue Large RNA Do not exceed the recommended amounts of starting Removal Column materials The amount of starting material may need to be has become decreased if the column shows clogging below the clogged recommended levels See also Clogged Column below acca It is recommended that the Elution Solution A supplied with this kit be used for maximum RNA recovery was used Different tissues and cells have different RNA contents Some tissues may not contain small RNA at detectable Low RNA content levels when processing the small sample sizes required for this procedure oe The flowthrough from the binding step with the Large RNA step was g Removal Column contains the small RNA molecules thus Poor RNA discarded ensure that
8. A was not washed three times with the provided Wash Traces of salt from the binding step may remain in the sample if the microRNA Enrichment Column is not washed three times with Wash Solution A Salt may interfere with downstream applications and thus must be perform well Solution A washed from the column in downstream applications Ensure that the dry spin under the Column Wash procedure is performed in order to remove traces of Elhanoheanyover ethanol prior to elution Ethanol is known to interfere with many downstream applications Genomic Large amount of DNA starting material Perform RNase free DNasel digestion on the RNA sample contamination used after elution to remove genomic DNA contamination Large RNA species present in elution Improper amount of ethanol added to the lysate before binding to the Large RNA Removal Column Ensure that the appropriate amount of ethanol was added to the lysate before it is applied to the Large RNA Removal Column This is imperative in order to capture the large RNA molecules onto the column Large amount of starting material used Repeat purification using less starting material Alternatively the isolation procedure can be repeated using the elution as the input The elution volume should first be adjusted to 300 uL using the provided Buffer RL The procedure can then be followed as written in the manual starting with the addition of ethanol
9. Benchtop microcentrifuge e mercaptoethanol Optional e 96 100 ethanol e RNase free microcentrifuge tubes For Animal Cell Protocol e PBS RNase free For Animal Tissue Protocol e Liquid nitrogen e Mortar and pestle e 25 gauge needle and syringe For Bacterial Protocol e Lysozyme containing TE Buffer o For Gram negative bacteria 1 mg mL lysozyme in TE Buffer o For Gram positive bacteria 3 mg mL lysozyme in TE Buffer For Plant Protocol e Liquid nitrogen e Mortar and pestle 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 sa
10. 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 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 There are 2 different spin columns provided with this kit the Large RNA Removal Column and the microRNA Enrichment Column Ensure that the correct column is used for each step of the procedure 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 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
11. ase 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 A few uL of media may be left behind with the pellet in order to ensure that the pellet is not dislodged Add 300 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 Add 150 uL of 96 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 1B Lysate Preparation from Animal Tissues Notes Prior to Use 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 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 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
12. ditional 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 at 70 C for long term storage Appendix A Optional Large RNA Purification Protocol 1 Column Wash a Reassemble the Large RNA Removal Column with the collection tube used in Step 2b b Apply 400 uL of Wash Solution A to the Large RNA Removal Column and centrifuge for 1 minute at 14 000 x g 14 000 RPM Note Ensure that 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 Discard the flowthrough and reassemble the spin column with the collection tube Repeat steps 1b and 1c to wash the column a second time e Wash column a third time by adding another 400 uL of Wash Solution A and centrifuging for 1 minute f Discard the flowthrough and reassemble the spin column with its collection tube g Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube a9 2 Large RNA Elution a Place the Large RNA Removal 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
13. ent in sample Removal Column Centrifuge Ensure that the centrifuge remains at room temperature g throughout the procedure Temperatures below 15 C may temperature too ae low 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 followed when working with contamination 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 Procedure not that the procedure be performed quickly This is Snormed auieki especially important for the Cell Lysate Preparation Step ee h q Y in the Animal Tissue protocol since the RNA in animal g tissues is not protected after harvesting until it is disrupted and homogenized RNA is Degraded Improper storage of the purified RNA 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 Frozen tissues or pellets were allowed to thaw prior to disruption Tissue samples should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage 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 12 Problem Possible Cause Solution and Explanation RNA does not RN
14. form The process involves the use of two different spin columns the Large RNA Removal Column and the microRNA Enrichment Column please see flow chart on page 3 Briefly the cells or tissues of interest are lysed using the provided Buffer RL and ethanol is then added to the sample The lysate is then applied to the Large RNA Removal Column and the larger RNA molecules will bind to the resin in the spin column while the smaller RNA species will pass through into the flowthrough Ethanol is then added to the flowthrough and the sample is applied to the microRNA Enrichment Column The small RNA molecules will then bind to the resin and any impurities are removed through a series of washes with the provided Wash Solution A The small RNA molecules are then eluted using the Elution Solution A and are ready for use in various applications Specifications Kit Specifications Maximum Column Binding Capacity 50 ug Maximum Column Loading Volume 650 uL Minimum Elution Volume 20 uL Size of RNA Purified lt 200 nt Maximum Amount of Starting Material Animal Cells 3 x 10 cells Animal Tissues 5 25 mg Bacteria 1 x 10 cells Plant Tissues 50 mg Blood 100 uL Time to Complete 10 Purifications 25 minutes Advantages e Fast and easy processing using rapid spin column format No phenol or chloroform extractions Isolate all small RNA molecules lt 200 nt Minimal contamination from large RNA molecules and genomic DNA
15. gn x NORGEN BIOTEK wi CORPORATION microRNA Purification Kit 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com Product Insert Product 21300 Introduction Norgen s microRNA Purification Kit provides a rapid method for the isolation and purification of small RNA molecules lt 200 nt from cultured animal cells small tissue samples bacterial cells plants and blood These small RNAs include regulatory RNA molecules such as microRNA miRNA and short interfering RNA siRNA as well as tRNA and 5S rRNA Small RNA molecules are often studied due to their ability to regulate gene expression miRNAs and siRNAs are typically 20 25 nucleotides long and regulate gene expression by binding to mRNA molecules and affecting their stability or translation The small RNA molecules isolated using Norgen s microRNA Purification Kit can be used in various downstream applications relating to gene regulation and functional analysis including RT PCR northern blotting and microarray analysis Norgen s Purification Technology Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix Norgen s resin binds RNA in a manner that depends on ionic concentrations The small RNA molecules are preferentially purified from other cellular components such as ribosomal RNA without the use of phenol or chloro
16. it is not inadvertently discarded Recovery Ethanol was not added to the flowthrough before binding to the microRNA Enrichment Column Ensure that the appropriate amount of ethanol was added to the flowthrough from the first binding step before it is applied to the microRNA Enrichment Column This is imperative in order to capture the small RNA molecules Ethanol was not added to the Wash Solution A Ensure that 90 mL of 96 100 ethanol is added to the supplied Wash Solution A prior to use 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 Bacteria All traces of media not removed Ensure that all media is removed prior to the addition of the Buffer RL through aspiration 11 Problem Possible Cause Solution and Explanation Insufficient Ensure that the appropriate amount of Lysis Buffer was solubilization of used for the amount of cells or tissue cells or tissues Maximum number of cells or amount Refer to specifications to determine if amount of starting of tissue exceeds material falls within kit specifications kit specifications Clogged Column High amounts of The lysate may be passed through a 25 gauge needle attached to a syringe 5 10 times in order to shear the genomic DNA genomic DNA prior to loading onto the Large RNA pres
17. l Strains Lysozyme Concentration i i Bacteria Type y Hh esa tl Incubation Time Gram negative 1 mg mL 5 min Gram positive 3 mg mL 10 min D Lysate Preparation from Plant Notes Prior to Use The maximum recommended input of plant tissue is 50 mg or 5 x 10 cells Both fresh and frozen plant tissues can be used for this protocol Samples should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage 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 It is important to work quickly during this procedure 1D Cell Lysate Preparation from Plants a Transfer lt 50 mg of plant tissue or 5 x 10 plant cells into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the sample into a fine powder using a pestle in liquid nitrogen Note If stored frozen samples are used do not allow the samples to thaw before transferring to the liquid nitrogen Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 600 uL of Buffer RL to the tissue sample and continue to grind until the sample has been homogenized Using a pipette transfer the lysate into an RNase free microcentrifuge tube not provided Spin the lysate for 2 minutes to pellet any cell debris Transfer the supernatant to another RNase free microce
18. mples ensure that they remain on ice during downstream applications Flow Chart Procedure for Purifying Small RNA Molecules using Norgen s microRNA Purification Kit Lyse cells or tissue using Buffer RL SPIN SPIN SPIN A TA au ODA cee A ca A Add Ethanol Bind large RNA molecules to Large RNA Removal Column Retain Flowthrough Add ethanol to flowthrough Bind small RNA molecules from flowthrough onto microRNA Enrichment Column Wash three times with Wash Solution A Elute small RNA with Elution Solution A Purified Small RNA miRNA siRNA tRNA 5S rRNA etc 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 rpm 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 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
19. ntrifuge tube Note the volume of the supernatant lysate Add a volume of 96 100 ethanol provided by the user that is equivalent to 50 of the lysate volume 50 uL of ethanol is added to every 100 uL of lysate Vortex to mix Proceed to Step 2 1E Lysate Preparation from Blood Notes Prior to Use 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 It is recommended that no more than 100 uL of blood be used in order to prevent clogging of the column We recommend the use of this kit to isolate RNA from non coagulating fresh blood using EDTA as the anti coagulant It is important to work quickly during this procedure 1E Cell Lysate Preparation from Blood a b Cc Transfer up to 100 uL of non coagulating blood to an RNase free microcentrifuge tube not provided Add 250 uL of Buffer RL to the blood Lyse cells by vortexing for 15 seconds Ensure that mixture becomes transparent before proceeding to the next step Add 150 uL of 96 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 Section 2 Purification of Small RNA from All Types of Lysate Note The remaining steps of the procedure for the purification of small RNA are the same from this point forward for all the different types of lysate 2 Large RNA
20. reparation from Bacteria Notes Prior to Use Prepare the appropriate lysozyme containing TE Buffer as indicated in Table 2 This solution should be prepared with sterile RNAse free TE Buffer and kept on ice until needed These reagents are to be provided by the user It is recommended that no more than 10 bacterial cells be used in this procedure Bacterial growth can be measured using a spectrophotometer As a general rule an E coli culture containing 1 x 10 cells mL has an ODs of 1 0 For RNA isolation bacteria should be harvested in log phase growth Bacterial pellets can be stored at 70 C for later use or used directly in this procedure Frozen bacterial pellets should not be thawed prior to beginning the protocol Add the Lysozyme containing TE Buffer directly to the frozen bacterial pellet Step 1Cc 1C Cell Lysate Preparation from Bacteria a b C Pellet bacteria by centrifuging at 14 000 x g 14 000 RPM for 1 minute Decant supernatant and carefully remove any remaining media by aspiration Resuspend the bacteria thoroughly in 100 uL of the appropriate lysozyme containing TE buffer see Table 2 by vortexing Incubate at room temperature for the time indicated in Table 2 Add 200 uL of Buffer RL and vortex vigorously for at least 10 seconds Add 150 uL of 96 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 Table 2 Incubation Time for Different Bacteria
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