Protocol (96-well) - Norgen Biotek Corp.
Contents
1. Ethanol is then added to the lysate and the solution is loaded onto the 96 Well Filter Plate Norgen s resin binds RNA in a manner that depends on ionic concentrations Thus only the RNA will bind to the resin in the wells 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 purified RNA is of the highest integrity and can be used in a number of downstream applications Specifications Kit Specifications Binding Capacity Per Well 50 ug Maximum Loading Volume Per Well 500 uL f es All sizes including small RNA Size of RNA Purified lt 200 nt Maximum Amount of Starting Material 7 Animal Cells 1 x 10 cells Animal Tissues 10 mg Blood 100 uL Plasma Serum 150 uL Bacteria 1 x 10 cells Yeast 1 x 10 cells Fungi 40 mg Plant Tissues 40 mg Time to Complete 96 Purifications 30 minutes Average Yields HeLa Cells 1 x 10 cells 15 ug E coli 1 x 10 cells 50 ug average yields will vary depending upon a number of factors including species growth conditions used and developmental stage Advantages e Fast and easy processing using either a vacuum manifold or centrifugation Isolate total RNA from large rRNA down to microRNA miRNA No phenol or chloroform extractions Isol2. 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 75 uL aliquot is required for each column to be treated Perform the appropriate Total RNA Isolation Procedure for your starting material up to and including Binding RNA to 96 Well Filter Plate Steps 1 and 2 of all protocols For Vacuum Manifold Apply 400 uL of Wash Solution A to each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or a pad provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes For Centrifugation Apply 400 uL of Wash Solution A to each well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes Discard the flowthrough Reassemble the 96 Well Filter Plate with the vacuum manifold or the bottom plate Apply 75 uL of the RNase free DNase solution prepared in Step 1 to each well of the 96 Well Filter Plate For Vacuum Manifold Apply vacuum for 30 seconds For Centrifugation Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 30 seconds After the centrifugation or vacuum in Step 5 pipette the flowthrough that is present in the collection plate back onto the top of the column Note Ensure Step
3. Reassemble the 96 Well Filter Plate and the bottom plate Repeat steps 3a and 3b to wash column for a second time Repeat steps 3a and 3b to wash column for a third time Pat the bottom of the 96 Well Filter Plate dry Reassemble the 96 Well Filter Plate and the bottom plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 5 minutes in order to completely dry the plate 29205 4 RNA Elution a Stack the 96 Well Filter Plate on top of the 96 Well Elution Plate b Add 75 uL of Elution Solution A to each well of the 96 Well Filter Plate c Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes 5 Storage of RNA Use the provided adhesive tape to seal the 96 Well Elution Plate 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 12 Appendix A Protocol for Optional On Column DNA Removal Norgen s Total RNA Purification 96 Well Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional protocol is provided below for maximum removal of residual DNA that may affect sensitive downstream applications It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step 1 For every on column reaction to be performed prepare a mix of 10 uL of DNase I and 65 uL of Enzyme Incubation Buffer using Norgen s RNase Free DNase Kit Product 25710
4. Wash Solution A This will give a final volume of 128 mL The labels on the bottles have a box that may be checked to indicate that the ethanol has been added e The volumes stated in each procedure for lysate preparation are the volumes required to prepare samples for each well of the 96 well plate 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 as well as for most plant tissues 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 The recommended input is 5 x 10 cells per well of the 96 Well Filter Plate However up to 1 x 10 cells may be processed for most cell lines A hemocytometer can be used in conjunction with a microscope to count the number of cells 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 Frozen pellets should be stored for no longer than 2 weeks to ensure that the integrity of the RNA is not compromised Frozen cell pellets should not
5. 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 96 Well Plate or other Multi Well Plate a 1A ii a9 Aspirate media and wash cell monolayer with an appropriate amount of PBS Aspirate PBS Add 300 uL of Buffer RL directly to each well of the multi well culture plate Lyse cells by gently tapping culture plate and swirling buffer around plate surface for two minutes Add 120 uL of 96 100 ethanol provided by the user to each well Mix by pipetting up and down a few times Cell Lysate Preparation from Cells Growing in Suspension and Lifted Cells Transfer cell suspension to the wells of an RNase free 96 well microplate not provided and centrifuge at no more than 200 x g 2 000 RPM for 10 minutes to pellet cells Aspirate supernatant carefully to ensure that the pellets are not dislodged Add 300 uL of Buffer RL to each well Lyse cells by gently tapping culture plate and swirling buffer around plate surface for two minutes Add 120 uL of 96 100 ethanol provided by the user to each well Mix by pipetting up and down a few times 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
6. gt k 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK amp CORPORATION Email techsupport norgenbiotek com Total RNA Purification 96 Well Kit Product Insert Product 24300 Norgen s Total RNA Purification 96 Well Kit provides a rapid method for the high throughput isolation and purification of total RNA from cultured animal cells tissue samples blood plasma serum bacteria yeast fungi and plants The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA 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 96 well 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 purification could be performed on either a vacuum manifold or using centrifugation The process involves first lysing the cells or tissue of interest with the provided Buffer RL please see the flow chart on page 4
7. the entire wash volume has not passed apply vacuum for an additional 2 minutes b Turn off vacuum and ventilate the manifold Discard the flowthrough c Reassemble the 96 Well Filter Plate and the vacuum manifold Repeat steps 3a and 3b to wash column for a second time d Reassemble the 96 Well Filter Plate and the vacuum manifold Repeat steps 3a and 3b to wash column for a third time e Pat the bottom of the 96 Well Filter Plate dry Reassemble the 96 Well Filter Plate and the vacuum manifold Apply vacuum for an additional 5 minutes in order to completely dry the plate f Turn off vacuum and ventilate the manifold 4 RNA Elution a Replace the collection waste tray in the vacuum manifold with the provided 96 Well Elution Plate Complete the vacuum manifold assembly with the 96 Well Filter Plate b Add 75 uL of Elution Solution A to each well of the plate c Apply vacuum for 2 minutes 5 Storage of RNA Use the provided adhesive tape to seal the 96 Well Elution Plate The purified RNA samples may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage B Total RNA Purification from All Types of Lysate Using Centrifugation Note The remaining steps of the procedure for the purification of total RNA using centrifugation are the same from this point forward for all the different types of lysate 2 Binding RNA to 96 Well Filter Plate a Place the 96 Well Filter Plate on top of
8. 2000 RPM for the RNA Binding Step is recommended Optional Step Norgen s Total RNA Purification Kit isolates total 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 It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step This step should be performed at this point in the protocol Use 96 100 Ethanol provided by the user instead of the provided Wash Solution A for the Wash Step 14 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 Wells of the plate materials The amount of starting material may need to have become be decreased if the wells of the plate show clogging clogged below the recommended levels See also Clogged Wells in Plate below pares Was It is recommended that the Elution Solution A supplied need 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 wells of the plate Fthanolwasiot Ensure that 90 mL of 96 100 ethanol is added to the added to t
9. 230 ratio will be lower lt 1 80 than the normal acceptable range from other cells or tissues Nonetheless these isolated RNA could still be used effectively in different downstream applications such as RT qPCR or microarrays 1 Cell Lysate Preparation from Plasma Serum a b Cc d Transfer up to 150 uL of plasma or serum to an RNase free microcentrifuge tube or an RNase free Deep Well 96 well microplate not provided Add 250 uL of Buffer RL to every 100 uL of plasma or serum Mix by vortexing for 10 seconds Optional Add 0 7 uL of 0 8 ug ul MS2 RNA per sample Note The addition of MS2 RNA could increase the consistency of RNA isolation Add 350 uL of 96 100 ethanol provided by the user to every 350 uL of the lysate equivalent to every 100 uL plasma or serum used Mix by agitation or by pipetting up and down a few times Proceed to Step 2 below 2 Purification of RNA using 96 Well Filter Plate Proceed to Section 2 for Total RNA Purification using either a vacuum manifold or centrifugation For purification using vacuum please follow the procedure outlined in Section 2A For purification using centrifugation please follow the procedure outlined in Section 2B For both protocols use 96 100 Ethanol provided by the user instead of the provided Wash Solution A for the Wash Step NOTE For higher recovery of small RNA species using the centrifugation protocol a lower centrifugation speed of 1500 x g
10. 6 is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species Incubate the assembly at 25 30 C for 15 minutes Without any further centrifugation proceed directly to RNA Wash Section 2A Step 3b for Vacuum Manifold procedure or Section 2B Step 3c for Centrifugation procedure 13 Appendix B Protocol for Total RNA Purification from Plasma or Serum Notes Prior to Use Plasma or serum 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 plasma or serum We recommend the use of this kit to isolate RNA from plasma or serum prepared by standard protocol from non coagulating fresh blood using EDTA or sodium citrate as the anti coagulant It is recommended that no more than 150 uL of plasma or serum be used in order to prevent clogging of the column Avoid multiple freeze thaw cycle of the plasma or serum sample Aliquot to the appropriate volume for usage prior to freezing Substitute the provided Wash Solution A with 96 100 Ethanol User provided It is important to work quickly during this procedure The yield of RNA from plasma and serum is highly variable In general the expected yield could vary from 1 to 100 ng per 100 uL plasma or serum used In addition the expected A260 A280 ratio as well as the A260 A
11. 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 homogenization The optimal amount of non fibrous tissue be used per well of the 96 Well Filter Plate is up to 8 mg However for most tissues except tissues with high cell number such as liver and spleen up to 10 mg could be processed For fibrous tissue such as heart a maximum of 2 mg is recommended 1B Cell Lysate Preparation from Animal Tissues a b C d Excise the tissue sample from the animal Determine the amount of tissue by weighing It is recommended that no more than 10 mg of tissue be used for each well of the 96 Well Filter Plate Transfer the tissue samples to appropriate vessels for the desired disruption method Add 350 uL of Buffer RL to each tissue sample Note Ensure that frozen tissues do not thaw during weighing or prior to the addition of Buffer RL For maximum RNA recovery homogenize frozen tissues to fine powder in liquid nitrogen prior to the addition of Buffer RL Homogenize the tissues using the appropriate cell disruption tool Note Thorough homogenization is required for optimal performance For tissue inputs of lt 8 mg it is not require
12. a provided 96 Well Collection Plate b Apply up to 500 uL of the lysate with the ethanol from Step 1 into each well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes Note Depending on the starting material a small quantity of precipitates may appear in the lysate ethanol mix No additional step is required to remove these precipitates prior to application to the wells c Discard the flowthrough Reassemble the the 96 Well Filter Plate and the bottom plate Note Ensure that all of the lysate from each well has passed through into the bottom plate If the entire lysate volume has not passed centrifuge for an additional 2 minutes 11 Optional Step Norgen s Total RNA Purification 96 Well Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional On Plate 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 3 RNA Wash a Apply 400 uL of Wash Solution A to each well of the 96 Well Filter Plate Centrifuge the assembly at maximum speed or 3 000 x g 3 000 RPM for 2 minutes Note Ensure the entire Wash Solution A has passed through into the bottom plate by inspecting the 96 Well Filter Plate If the entire wash volume has not passed centrifuge for an additional 2 minutes Discard the flowthrough
13. ate high quality total RNA from a variety of sources RNA can be isolated and detected from as little as a single animal cell Kit Components Component Product 24300 192 preps Buffer RL 2x40 mL Wash Solution A 2x 38 mL Elution Solution A 2x 20mL 96 Well Filter Plate 2 Adhesive Tape 96 Well Collection Plate 96 Well Elution Plate Product Insert P P s 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 Su
14. ay Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2012 Norgen Biotek Corp P124300 12 M14 17
15. d to perform centrifugation to remove cell debris if the homogenization is complete For tissue inputs larger than 8 mg or if incomplete cell disruption is suspected centrifuge the lysate at maximum speed for 2 minutes in an appropriate centrifuge Transfer the supernatant to a new 96 well microplate Add 120 uL of 96 100 ethanol provided by the user to each tissue sample Mix by pipetting up and down a few times 1C Lysate Preparation from Blood Notes Prior to Use This procedure is for the isolation of RNA from whole blood For the isolation of RNA from plasma or serum samples please see Appendix B 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 per well of the 96 Well Filter Plate in order to prevent clogging of the plate 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 1C Cell Lysate Preparation from Blood Transfer up to 100 uL of non coagulating blood to each well in an RNase free 96 well microplate not provided Add 200 uL of Buffer RL Lyse cells by gently tapping the 96 well microplate and swirling buffer around plate surface for two minutes Add 120 uL of 96 100 ethanol
16. e remains at room temperature throughout the procedure Temperatures below 15 C may cause precipitates to form that can cause the wells to clog RNA is Degraded RNase contamination 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 Procedure not performed quickly enough In order to maintain the integrity of the RNA it is important that the procedure be performed quickly This is especially important for the Cell Lysate Preparation Step in the Animal Tissue protocol since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized 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 cell pellets were allowed to thaw prior to RNA isolation Do not allow frozen tissues to thaw prior to isolation 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 Lysozyme or lyticase used may not be RNAse free Ensure that the lysozyme and lyticase being used with this kit is RNase free in order to pr
17. er RL For maximum RNA recovery homogenize frozen tissues to fine powder in liquid nitrogen prior to the addition of Buffer RL Homogenize the tissues using the appropriate cell disruption tool Centrifuge the lysate at maximum speed for 2 minutes in an appropriate centrifuge Transfer the supernatant to a new 96 well microplate Add 120 uL of 96 100 ethanol provided by the user to each lysate sample Mix by pipetting up and down a few times 1G Lysate Preparation from Plant Notes Prior to Use The maximum recommended input of plant tissue is 40 mg or 5 x 10 plant cells per well of the 96 Well Filter Plate Both fresh and frozen plant samples 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 1G Cell Lysate Preparation from Plant a Excise the tissue sample from the plant b Determine the amount of tissue by weighing It is recommended that no more than 40 mg of tissue be used per well of the 96 Well Filter Plate c Transfer the tissue samples to appropriate vessels for the desired disruption method d Add 350 uL of Buffer RL to each tissue sample Note Ensure that frozen tissues do not thaw during weighing or prio
18. event possible problems with RNA degradation RNA does not RNA was not washed 3 times with the provided Traces of salt from the binding step may remain in the sample if the plate is not washed 3 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 vacuum or dry spin under the RNA applications Wash procedure is performed in order to remove traces Ethanol carryover i of ethanol prior to elution Ethanol is known to interfere with many downstream applications Genomic Large amounts of Perform RNAse free DNasel digestion on the RNA DNA starting material sample after elution to remove genomic DNA contamination used contamination 16 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 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 Parkw
19. he Wash lied Wash Solution A prior t Poor RNA Solution A supplied Wash Solution A prior to use Recovery Different tissues and cells have different RNA contents Low RNA content and thus the expected yield of RNA will vary greatly from in cells or tissues these different sources Please check literature to used determine the expected RNA content of your starting material Cell Culture Cell Ensure that the cell monolayer is washed with the monolayer was not appropriate amount of PBS in order to remove residual washed with PBS media from cells Yeast Lyticase was not added to Ensure that the appropriate amount of Lyticase is added the Resuspension when making the Resuspension Buffer Buffer Bacteria and veast Ensure that all media is removed prior to the addition of All traces of media ei the Buffer RL through aspiration not removed Insufficient Ensure that the appropriate amount of lysis buffer was solubilization of used for the amount of cells or tissue cells or tissues Clogged Wells in Plate 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 15 Problem Possible Cause Solution and Explanation RA Ensure that a vacuum pressure of at least 650 mbar or Insufficient Vacuum 25 in Hg is developed Clogged Wells in Plate Centrifuge temperature too low Ensure that the centrifug
20. m temperature The correct pressure can be calculated using the conversions 1 mbar 100 Pa 0 0394 in Hg 0 75 mm Hg 0 0145 psi For Centrifugation 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 with the exception of the protocol for plasma serum A separate protocol for the isolation of total RNA from plasma serum samples is located in Appendix B e Please ensure that the correct procedure for preparing the lysate from your starting material is followed as indicated in the table below Sample Type Lysate Preparation Page Cultured Cells 6 Animal Tissue 6 Blood 7 Plasma Serum 14 Bacteria 7 Yeast 8 Fungi 9 Plant 9 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 bottles containing the concentrated
21. p and down a few times Add 120 uL of 96 100 ethanol provided by the user to lysate Mix by pipetting up and down a few times Table 1 Incubation Time for Different Bacterial Strains Lysozyme Concentration Incubation Time Sa ETE NIRS in TE Bufffer Gram negative 1 mg mL 5 min Gram positive 3 mg mL 10 min 1E Lysate Preparation from Yeast Notes Prior to Use Prepare the appropriate amount of Lyticase containing Resuspension Buffer considering that 75 uL of buffer is required for each preparation The Resuspension Buffer should have the following composition 50 mM Tris pH 7 5 10 mM EDTA 1M Sorbital 0 1 B mercaptoethanol and 1 unit uL Lyticase This solution should be prepared with sterile RNAse free reagents and kept on ice until needed These reagents are to be provided by the user It is recommended that no more than 10 yeast cells or 1 mL of culture be used per well of the 96 Well Filter Plate For RNA isolation yeast should be harvested in log phase growth Yeast can be stored at 70 C for later use or used directly in this procedure Frozen yeast pellets should not be thawed prior to beginning the protocol Add the Lyticase containing Resuspension Buffer directly to the frozen yeast pellet Step 1Ec 1E Cell Lysate Preparation a b Pellet yeast by centrifuging at 14 000 x g 14 000 RPM for 1 minute for culture collected in 1 5 mL microfuge tubes or 3000 x g 3 000 RPM fo
22. pplied Reagents and Equipment You must have the following in order to use the Total RNA Purification 96 Well Kit For All Protocols e For Vacuum Format o Vacuum manifold with vacuum pump capable of generating a minimum pressure of 650 mbar or 25 in Hg such as Whatman UniVac 3 Vacuum to Collect Manifold o Sealing tape or pads e For Centrifuge Format o Centrifuge with rotor for 96 well plate assembly such as Thermo Fisher IEC Centra CL3 series or Beckman GS 15R e 96 100 ethanol e f mercaptoethanol optional e Collection Waste Tray for vacuum manifold or 96 well bottom plate single or 96 well format for centrifugation Two 96 Well Collection Plates are provided with the kit For Animal Cell Protocol e PBS RNase free For Animal Tissue Protocol e Liquid nitrogen e Cell Disruption Tool such as mortar and pestle rotor stator homogenizer or bead mills 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 Yeast Protocol e Resuspension Buffer with Lyticase o 50mM Tris pH 7 5 o 10 mM EDTA o 1 M Sorbital o 1 unit uL Lyticase For Fungi Protocol e Liquid nitrogen e Cell Disruption Tool such as mortar and pestle rotor stator homogenizer or bead mills For Plant Protocol e Liquid nitrogen e Cell Disruption Tool such as mortar and pestle rotor stator homogenizer or bead mills For Plasma Serum Pro
23. provided by the user to each well Mix by pipetting up and down a few times 1D Lysate Preparation from Bacteria Notes Prior to Use Prepare the appropriate lysozyme containing TE Buffer as indicated in Table 1 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 per well for 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 1Dc 1D Cell Lysate Preparation from Bacteria a Pellet bacteria by centrifuging at 14 000 x g 14 000 RPM for 1 minute for culture collected in 1 5 mL microfuge tubes or 3000 x g 3 000 RPM for 5 minutes for culture in a 96 well microplate Carefully remove media by aspiration Resuspend each bacterial pellet thoroughly in 75 uL of the appropriate lysozyme containing TE buffer see Table 1 Incubate at room temperature for the time indicated in Table 1 Add 225 uL of Buffer RL to each bacteria sample Mix by pipetting u
24. r 5 minutes for culture in a 96 well microplate Carefully remove media by aspiration Resuspend the yeast pellets thoroughly in 75 uL of Lyticase containing Resuspension Buffer Incubate at 37 C for 10 minutes Add 225 uL of Buffer RL to each yeast sample Mix by pipetting up and down a few times Add 120 uL of 96 100 ethanol provided by the user to the lysate Mix by pipetting up and down a few times 1F Lysate Preparation from Fungi Notes Prior to Use Fresh or frozen fungi may be used for this procedure Fungal tissues should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Fungi 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 It is recommended that no more than 40 mg of fungi be used per well of the 96 Well Filter Plate to prevent clogging of the plate 1F Cell Lysate Preparation from Fungi a b c d Excise the tissue sample from the fungus Determine the amount of tissue by weighing It is recommended that no more than 50 mg of tissue be used per well of the 96 Well Filter Plate Transfer the tissue samples to appropriate vessels for the desired disruption method Add 350 uL of Buffer RL to each tissue sample Note Ensure that frozen tissues do not thaw during weighing or prior to the addition of Buff
25. r any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes Note Depending on the starting material a small quantity of precipitates may appear in the lysate ethanol mix No additional step is required to remove these precipitates prior to application of the mixture to the wells c Turn off vacuum and ventilate the manifold Discard the flowthrough Reassemble the 96 Well Filter Plate and the vacuum manifold Note Ensure that all of the lysate from each well has passed through into the collection waste tray If the entire lysate volume has not passed apply vacuum for an additional 2 minutes Optional Step Norgen s Total RNA Purification 96 Well Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional On Plate 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 10 3 RNA Wash a Apply 400 uL of Wash Solution A to each well of the 96 Well Filter Plate Tape the plate or any unused wells using sealing tape or pads provided by the user according to the vacuum manifold manufacturer s recommendations Apply vacuum for 2 minutes Note Ensure the entire Wash Solution A has passed through into the collection waste tray by inspecting the 96 Well Filter Plate If
26. r to the addition of Buffer RL For maximum RNA recovery homogenize frozen tissues to fine powder in liquid nitrogen prior to the addition of Buffer RL e Homogenize the tissues using the appropriate cell disruption tool Centrifuge the lysate at maximum speed for 2 minutes in an appropriate centrifuge Transfer the supernatant to a new 96 well microplate g Add 120 uL of 96 100 ethanol provided by the user Mix by pipetting up and down a few times Section 2 Total RNA Purification from All Types of Lysate Note The purification of total RNA from the lysate prepared in Section 1 could be performed using either a vacuum manifold or centrifugation For purification using vacuum please follow the procedure outlined in Section 2A For purification using centrifugation please follow the procedure outlined in Section 2B A Total RNA Purification from All Types of Lysate Using Vacuum Manifold Note The remaining steps of the procedure for the purification of total RNA using a vacuum manifold are the same from this point forward for all the different types of lysate 2 Binding RNA to 96 Well Filter Plate a Assemble the 96 Well Filter Plate and the vacuum manifold according to manufacturer s recommendations Note The provided 96 Well Collection Plate can be used as the collection waste tray if desired b Apply up to 500 uL of the lysate with the ethanol from Step 1 into each well of the 96 Well Filter Plate Tape the plate o
27. tocol e MS2 RNA 0 8 yg ul Roche Cat No 10165948001 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 Flowchart Procedure for Purifying Total RNA using Norgen s Total RNA Purification 96 Well Kit Sample of cells or tissues Purified Total RNA Lyse cells or tissues with Buffer RL Add ethanol Bind RNA Wash three times with Wash Solution A Elute RNA with Elution Solution A Procedures For Vacuum Manifold All vacuum steps are performed at roo
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