Animal Tissue RNA Purification Kit Product Insert
Contents
1. 1 minute Note Ensure that the entire 115 uL of DNase mix passes through the column If needed spin at 14 000 x g 14 000 RPM for an additional minute b After the centrifugation in Step 3a pipette the flowthrough that is present in the collection tube back onto the top of the column Note Ensure Step 3b is performed in order to ensure maximum DNase activity and to obtain maximum yield of RNA in particular for small RNA species c Incubate at room temperature for 15 minutes 4 Column Wash a Apply 400 uL of Wash Solution A to the column containing the DNase mix 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 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 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 1 minute 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 RNA2. 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 Procedurenot important that the procedure be performed quickly This is especially important for the Cell Lysate Preparation performed quickly Step in th tocol si the RNA i imal ti l enough ep in the protocol since the RNA in animal 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 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 Problem Possible Cause Solution and Explanation RNA does not RNA was not washed 2 times with the provided Traces of salt from the binding step may remain in the sample if the column is not washed 2 times with Wash Solution A Salt may interfere with downstream perform well Wash Solution A applications and thus must be washed from the column in downstream Ensure that the dry spin under the Column Wash applications Ethano
3. ade 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 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 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 that all solutions are at room temperature prior to use e All enzymes provided should remain at the storage temperature indicated on each vial until use e Reconstitute each of the provided Proteinase K vials in 600 uL of molecular biology grade w
4. ate at 55 C for 15 minutes Vortex the tubes occasionally during incubation Spin the lysate for 1 minute to pellet any cell debris Transfer the supernatant to a new RNase free microcentrifuge tube not provided Add 450 uL of 96 100 ethanol provided by the user to the lysate Vortex to mix 2 Binding RNA to Column a Assemble a column with one of the provided collection tubes b Apply up to 650 uL of the lysate with the 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 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 Note If part of the lysate has not passed into the collection tube after Step 2d and the volume is less than 200 uL continue to Step 2e without additional centrifugation e Apply 400 uL of Wash Solution A to the column and centrifuge for 2 minutes f Discard the flowthrough and assemble the spin column with a new collection tube 3 On Column DNAse Treatment Optional If DNase Treatment is not required proceed directly to Step 4a of Column Wash a Apply 100 uL of Enzyme Incubation Buffer A and 15 uL of DNase I to the column and centrifuge at 14 000 x g 14 000 RPM for
5. ater or 10 mM Tris HCl pH 7 5 RNase Free aliquot into small fractions and store the unused portions at 20 C until needed 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 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 1 Cell ze 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 RNA ate are compatible with this isolation procedure Prior to isolation carefully remove the tissue from the storage reagent using forceps and dry any excessive liquid It is very important no
6. e following in order to use the Animal Tissue RNA Purification Kit Benchtop microcentrifuge 96 100 ethanol B mercaptoethanol Liquid nitrogen Mortar and pestle 55 C incubator Flowchart Procedure for Purifying Total RNA using Norgen s Animal Tissue RNA Purification Kit Excise tissue sample Add Buffer RL and homogenize Add Proteinase K and incubate SPIN Add Ethanol Bind to column Wash once with Wash Solution A SPIN Add DNasel and incubate SPIN TA 4 am eA tt cana Wash twice with Wash Solution A SPIN A HL Elute RNA with Elution Solution A SPIN e _ Purified Total RNA 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 gr
7. eatment with the provided Proteinase K please see the flow chart on page 4 The sample is then centrifuged ethanol is added to the supernatant and the solution is loaded onto a 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 any remaining proteins and other contaminants will be removed in the flowthrough or retained on the top of the resin At this point an on column DNAase digestion is performed to remove any traces of DNA which may co purify with the RNA A series of wash steps are then performed to remove the DNase and any remaining impurities and lastly the purified total RNA is eluted with Elution Solution A 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 50 ug Maximum Column Loading Volume 650 uL Las All sizes including small RNA Size of RNA Purified lt 200 nt Maximum Amount of Starting Material Heart 30 mg Kidney 15 mg Liver 15 mg Muscle 30 mg Spleen 15 mg Time to Complete 10 Purifications 50 minutes Average Yields Rat Muslce 10 mg 5 ug Rat Liver 10 mg 30 pg Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature The DNAse should be stored at 20 C upon arrival The Proteinase K should be stored at 20 C upon ar
8. g DR NORGEN BIOTEK wie CORPORATION 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com Animal Tissue RNA Purification Kit Product Insert Product 25700 Norgen s Animal Tissue RNA Purification Kit provides a rapid method for the isolation and purification of total RNA from all types of animal tissue samples including fiber rich tissues such as muscle and heart Norgen s Animal Tissue RNA Purification Kit is provided with Proteinase K which aids in the removal of the various proteins present in fiber rich tissues including collagen contractile proteins and connective tissues The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA without the use of inhibitory 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 tissue of interest with Buffer RL followed by tr
9. l carryover procedure is performed in order to remove traces of ethanol prior to elution Ethanol is known to interfere with many downstream applications Large amounts of Perform the RNAse free DNasel digestion on the RNA starting material sample suggested in the protocol to remove genomic used DNA contamination Genomic DNA contamination DNase mix did not completely pass through the column during DNase treatment Ensure the entire 115 uL of DNase mix passes through the column If needed spin at 14 000 x g 14 000 rpm for 1 minute Related Products Product RNase Free DNase Kit 25710 Total RNA Purification Kit 17200 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 P125700 8 M14
10. 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 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 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 ce wis It is recommended that the Elution Solution A supplied sed 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 was not 3 Recovery added to the Wash Ensure that 90 mL of 96 100 ethanol is added to the Solution A supplied Wash Solution A prior to use Low RNA content in tissue used Different tissues have different RNA contents and thus the expected yield of RNA will vary greatly from these different sources Please check literature to determine the expected RNA content of your starting material Insufficient solubilization of
11. rival and after reconstitution These reagents should remain stable for at least 1 year in their unopened containers Advantages e Isolate high quality total RNA from a variety of animal tissues including fiber rich tissues Isolate total RNA from large rRNA down to microRNA miRNA No phenol or chloroform extractions Fast and easy processing using rapid spin column format On column DNAse treatment for removal of any contaminating DNA Kit Components Component Product 25700 50 preps Buffer RL 30 mL RNase Free Water 40 mL Wash Solution A 38 mL Enzyme Incubation Buffer A 6 mL Elution Solution A 6 mL Proteinase K 2 vials DNase 1 vial Mini Spin Columns 50 Collection Tubes 100 Elution tubes 1 7 mL 50 Product Insert 1 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 Tissue 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 You must have th
12. t to exceed the recommended starting amount for each tissue Please refer to Table 1 below for the recommended maximum input amounts of each tissue Table 1 Recommended Maximum Input Amounts of Different Tissues Tissue Maximum Input Amount Heart 30 mg Kidney 15 mg Liver 15 mg Muscle 30 mg Spleen 15 mg Lysate Preparation 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 Note The use of liquid nitrogen is recommended However if homogenization without flash freezing is preferred proceed to Step 1e 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 Add 600 uL of RNase Free Water provided to the lysate Vortex to mix Add 20 uL of reconstituted Proteinase K to the lysate and incub
13. tissues Ensure the lysate is diluted with the appropriate amount of RNase free water and that the appropriate amount of Proteinase K is added Also ensure that the Proteinase K treatment is performed at 55 C for the full 15 minutes The incubation time can be increased up to 30 minutes if required Problem Possible Cause Solution and Explanation Ensure the lysate is diluted with the appropriate amount Insufficient of RNase free water and that the appropriate amount of solubilization of Proteinase K is added Also ensure that the Proteinase tissies K treatment is performed at 55 C for the full 15 minutes The incubation time can be increased up to 30 minutes if required Maxime amount Refer to specifications to determine if amount of starting of tissue exceeds t s PRE eat as material falls within kit specifications Clogged kit specifications Column Clogged Column The lysate may be passed through a 25 gauge needle High amounts of attached to a syringe 5 10 times in order to shear the genomic DNA genomic DNA prior to loading onto the column Also present in sample ensure that the on column DNase treatment is performed if high amounts of genomic DNA are found in the sample Centiifuge Ensure that the centrifuge remains at room temperature g throughout the procedure Temperatures below 15 C temperature too wie f h h iow may cause precipitates to form that can cause the columns to clog RNases may be
Download Pdf Manuals
Related Search