RiboMinus RNA - Thermo Fisher Scientific
Contents
1. 3 Centrifuge the cartridge at maximum speed for 1 minute at room temperature The Recovery tube contains purified and concentrated RiboMinus RNA sample that is depleted of rRNA 4 If you performed elution with 50 ul water perform a second elution step using 50 ul Sterile RNase free water pH gt 7 0 if desired The Recovery tube contains purified RiboMinus RNA Remove and discard the cartridge Based on the volume of elution buffer used for elution the recovery of the elution volume will vary and is usually 90 of the elution buffer volume used 5 Store RiboMinus RNA at 80 C or use RiboMinus RNA for the desired downstream application You may determine the quality and quantity of the purified RiboMinus RNA as described on the next page If the RiboMinus RNA sample shows considerable contamination from genomic DNA after analysis perform DNase I digestion on the purified sample Analyzing RiboMinus RNA RNA Yield The quantity of the purified RiboMinus RNA is easily quantitated using UV absorbance at 260 nm or Quant iT RNA Assay Kit UV Absorbance 1 Dilute an aliquot of the small sample in 10 mM Tris HCl pH 7 0 Mix well Transfer to a cuvette 1 cm path length Note The RNA must be in a neutral pH buffer to accurately measure the UV absorbance 2 Determine the OD of the solution using a spectrophotometer blanked against 10 mM Tris HCl pH 7 0 Calculate the amount of total2. as drying reduces the bead efficiency To aspirate the supernatant after bead washing place the pipette tip at the opposite side of the tube away from the beads Carefully remove the supernatant without disturbing or removing any beads Do not submerge the magnetic stand in water To clean the magnetic stand spray the stand with ethanol and wipe it with a paper towel Binding The binding capacity of RiboMinus Magnetic Beads is gt 3400 Capacity pmoles free biotin per mg of beads 10 Continued on next page Selective Hybridization and Removal of rRNA Continued Hybridization Step Preparing RiboMinus Magnetic Beads Instructions are provided below to perform hybridization for 10 ug of your total RNA sample with the RiboMinus Human Mouse Probe If you wish to use gt 10 ug total RNA sample adjust the volume of reagents accordingly and you may need to optimize the amount of beads needed for complete removal of rRNA 1 Seta water bath or heat block to 70 75 C 2 To a sterile RNase free 1 5 ml microcentrifuge tube add the following Total RNA 2 10 pg 20 ul RiboMinus Probe 100 pmol l 8 ul Hybridization Buffer B5 300 pl 3 Incubate the tube at 70 75 C for 5 minutes to denature RNA 4 Allow the sample to cool to 37 C slowly over a period of 30 minutes by placing the tube in a 37 C water bath To promote sequence specific hybridization it is important to allow slow cooling Avoid cooli
3. 600 ul 96 100 ethanol to obtain a total volume of 1 6 ml Mix well by vortexing Remove a Spin Cartridge in a Collection Tube from the package Load 700 ul sample containing Binding Buffer L3 and ethanol to the cartridge Centrifuge the cartridge at 12 000 x g for 1 minute at room temperature Discard the flow through Perform the loading step twice to load the remaining sample from Step 1 onto the cartridge and centrifuge the cartridge at 12 000 x g for 1 minute at room temperature Discard the flow through Proceed to Washing Step next page Continued on next page 15 Concentrating RiboMinus RNA Fraction Continued Washing Step Elution Step The Next Step 16 1 Add 600 ul Wash Buffer W5 with ethanol page 15 to the cartridge 2 Centrifuge the cartridge at 12 000 x g for 1 minute at room temperature Discard the flow through 3 Repeat the wash step with 600 ul Wash Buffer W5 with ethanol 4 Discard the collection tube and place the cartridge into a clean Wash Tube supplied with the kit 5 Centrifuge the cartridge at maximum speed for 2 3 minutes at room temperature to remove any residual Wash Buffer W5 Discard the Wash Tube 6 Proceed to Elution Step below 1 Place the Spin Cartridge in a clean 1 7 ml Recovery Tube supplied with the kit 2 Add 50 100 ul of Sterile RNase free water pH gt 7 0 to the center of the column Incubate the cartridge at room temperature for 1 minute
4. the hybridized sample from Step 4 previous page has cooled to 37 C briefly centrifuge the tube to collect the sample to the bottom of the tube Transfer the sample 328 ul to the prepared RiboMinus Magnetic beads from Step 8 above Mix well by inverting the tube repeatedly Incubate the tube at 37 C for 15 minutes During incubation gently mix the contents occasionally Place the tube on a magnetic stand for 1 minute to pellet the rRNA probe complex Do not discard the supernatant The supernatant contains RiboMinus RNA Transfer the supernatant 528 ul to a tube capable of holding 3X volume of the supernatant Resuspend beads in 50 ul Hybridization Buffer B5 to wash beads Place the tube on a magnetic stand for 1 minute Transfer the supernatant to the tube from Step 6 to obtain a total supernatant volume of 575 ul Proceed immediately to the Binding Step page 15 Concentrating RiboMinus RNA Fraction Introduction Elution Volume The concentration step is designed for concentrating RiboMinus RNA using spin column based centrifugation in a total time of 10 15 minutes See next page for an experimental outline The RiboMinus Human Mouse Transcriptome Isolation Kit buffer contains guanidine isothiocyanate Always wear a laboratory coat disposable gloves and eye protection when handling buffers Do not add bleach or acidic solutions directly to solutions containing guanidine isothiocyanate or sam
5. 15 minutes During incubation gently mix the contents occasionally Place the tube on a magnetic stand for 1 minute to pellet the rRNA probe complex The supernatant contains the RiboMinus RNA fraction Transfer the supernatant 528 ul to a tube capable of holding 3X volume of the supernatant Resuspend beads in 50 ul Hybridization Buffer B5 to wash beads Place the tube on a magnetic stand for 1 minute Transfer the supernatant to the tube from Step 4 to obtain a total supernatant volume of 575 ul Concentrating 1 To the sample from Step 5 above add 500 ul Binding RiboMinus Buffer L3 and 600 ul 96 100 ethanol Mix well RNA Bind 700 ul sample from Step 1 containing Binding Buffer L3 and ethanol to the spin cartridge Centrifuge the cartridge at 12 000 x g for 1 minute at room temperature Discard the flow through Perform this binding step twice to bind the remaining sample from Step 1 onto the cartridge and centrifuge the cartridge at 12 000 x g for 1 minute at room temperature Discard the flow through Wash the cartridge with 600 ul Wash Buffer W5 with ethanol page 15 Centrifuge the cartridge at 12 000 x g for 1 minute at room temperature Discard the flow through Repeat the wash step once Discard the tube and place the column into a clean Wash Tube supplied with the kit Centrifuge the cartridge at maximum speed for 2 3 minutes at room temperature to remove any residual Wash Buffer W5 Pl
6. A Total RNA Isolation Checking the Total RNA Quality You will need to isolate high quality total RNA from cells or tissues using a method of choice prior to using this kit To obtain high quality total RNA follow the guidelines recommended below Observe the following guidelines to prevent RNase contamination e Use disposable individually wrapped sterile plasticware e Use only sterile new pipette tips and microcentrifuge tubes e Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin e Always use proper microbiological aseptic techniques when working with RNA e Use RNase AWAY Reagent page viii to remove RNase contamination from surfaces Total RNA can be isolated from tissue or cells using the method of choice We recommend isolating total RNA using the Micro to Midi Total RNA Purification System or TRIzol Reagent available from Invitrogen page viii for ordering information You will use 2 10 ug total RNA for each reaction Resuspend isolated total RNA in DEPC treated water at a concentration of 0 5 ug ml Check the quality of your total RNA see below Store your total RNA at 80 C and avoid repeated freezing and thawing of total RNA To check total RNA integrity analyze 0 5 ug of your RNA by agarose ethidium bromide gel electrophoresis You should see the following on an agarose gel e 28S rRNA band 5 0 kb for human 4 7 kb for mou
7. NA sample with the Hybridization RiboMinus Human Mouse Probe as below 1 To a sterile RNase free 1 5 ml microcentrifuge tube add Total RNA 2 10 ug 20 pl RiboMinus Probe 100 pmol l 8 ul Hybridization Buffer B5 300 pl Incubate the tube at 70 75 C for 5 minutes to denature RNA Allow the sample to cool to 37 C slowly over a period of 30 minutes by placing the tube in a 37 C water bath Proceed to preparing beads below Preparing 1 Resuspend the RiboMinus Magnetic Beads in its bottle RiboMinus Pipet 500 ul of the bead suspension into a sterile RNase Magnetic Beads free 1 5 ml microcentrifuge tube Place the tube with the bead suspension on a magnetic stand for 1 minute Gently aspirate and discard the supernatant Add 500 ul sterile RNase free water to the beads and resuspend beads Place the tube on a magnetic stand for 1 minute Gently aspirate and discard the supernatant Repeat Step 4 once Resuspend beads in 500 ul Hybridization Buffer B5 Place the tube on a magnetic stand for 1 minute Gently aspirate and discard the supernatant Resuspend beads in 200 ul Hybridization Buffer B5 and keep the beads at 37 C until use Continued on next page iv Experienced Users Procedure Continued Removing Transfer 328 ul of the cooled hybridized sample from rRNA Step 3 previous page to the prepared RiboMinus Magnetic beads from Step 7 previous page and mix well Incubate the tube at 37 C for
8. RNA tRNA small rRNAs 5S rRNA 5 85 rRNA and may also contain regulatory RNA molecules such as microRNA miRNA and short interfering RNA siRNA snRNA and other RNA transcripts of yet unknown function The RiboMinus RNA molecules are part of the transcriptome and are important in protein coding signaling structural support of subcellular elements and transcriptional post transcriptional regulation The transcriptome is defined as the complete collection of transcribed elements of the genome Ruan et al 2004 and contains mRNA transcripts and non mRNA transcripts including RiboMinus RNA Transcriptome analysis is gaining increased attention in gene expression analysis Since large rRNA constitutes 90 95 RNA species in total RNA whole transcriptome analysis without any contamination from rRNA is very difficult and suggests the need for developing procedures for transcriptome isolation Current methods for RNA purification do not allow for efficient isolation of transcriptome The total RNA purification methods result in enriching the large rRNA molecules while the mRNA purification methods use polyA selection and or cap binding approaches that do not enrich for the complete transcriptome The RiboMinus Human Mouse Transcriptome Isolation Kit is a novel method of isolating transcriptome and involves selective removal of large rRNA from total RNA The isolated transcriptome is gt 95 depleted in rRNA and is enriched in al
9. RNA using the following formula Total RNA ug OD2 60 x 40 ug 1 OD260 x 1 ml x dilution factor x total sample volume ml The typical yield of RNA using the RiboMinus Human Mouse Transcriptome Isolation Kit is 1 ug RNA from 10 pg total RNA sample Quant iT RNA Assay Kits The Quant iI RNA Assay Kit page viii for ordering information provides a rapid sensitive and specific method for RNA quantitation with minimal interference from DNA protein or other common contaminants that affect UV absorbance readings The kit contains a state of the art quantitation reagent and pre diluted standards for standard curve The assay is performed in a microtiter plate format and is designed for reading in standard fluorescent microplate readers Continued on next page 17 Analyzing RiboMinus RNA Continued RNA Quality 18 The RNA isolated using the RiboMinus Human Mouse Transcriptome Isolation Kit is of high quality and is gt 95 depleted in rRNA species To verify rRNA depletion perform agarose gel electrophoresis of the sample Agarose gel electrophoresis analysis shows depletion of 18S and 28S rRNA bands as compared to a control sample see below for an example Absence of contaminating DNA and RNA degradation may be confirmed by agarose gel electrophoresis The efficiency of rRNA depletion can also be analyzed using a bioanalyzer such as the Agilent 2100 bioanalyzer using an RNA LabChip The bioanalyzer da
10. Recovery Tubes 1 7 ml Continued on next page vi Kit Contents and Storage Continued Product Qualification Limited Use Label License No 237 LNA Oligonucleotides The RiboMinus Human Mouse Transcriptome Isolation Kit is functionally qualified as described below Purified total RNA 10 ug from HeLa cells is subjected to ribosomal RNA depletion using the kit as described in this manual Agarose gel electrophoresis must show gt 95 depletion of 18S and 285 ribosomal bands from the purified sample as compared to control sample Bioanalyzer analysis of the purified sample must show lt 5 of peak area for 185 and 28S ribosomal bands as compared to peak area for the control sample RiboMinus Magnetic Beads The binding capacity of the beads must be gt 3400 pmoles free biotin per mg of streptavidin coated magnetic beads and must be free from bacterial contamination RiboMinus Human Mouse Probe The probes must contain the correct sequence and the locked nucleic acid LNA at the specified position for each probe Mass spectrometry analysis of probes must indicate the specified mass and HPLC analysis must indicate gt 85 purity The probes must be RNase and DNase free LNA oligonucleotides are produced under a license from Exiqon A S vii Accessory Products Additional The following products are also available from Invitrogen Products For more details on these products visit our Web site at
11. ace the cartridge in a 1 7 ml Recovery Tube Elute with 50 100 ul of Sterile RNase free water pH gt 7 0 Incubate the cartridge at room temperature for 1 minute Centrifuge the cartridge at maximum speed for 1 minute The Recovery Tube contains purified RiboMinus RNA Store RiboMinus RNA at 80 C or use RiboMinus RNA for the desired downstream application Kit Contents and Storage Shipping and All components of the RiboMinus Human Mouse Storage Transcriptome Isolation Kit are shipped at room temperature Upon receipt store the modules as follows e RiboMinus Human Mouse Module at 4 C e RiboMinus Concentration Module at room temperature Sufficient reagents are provided in the kit to perform 6 reactions RiboMinus The components included in the RiboMinus Human Mouse Human Mouse Module are listed below Module Store the module at 4 C For long term storage store the probe at 20 C RiboMinus Magnetic Beads 12 mg ml in Phosphate Buffered 3 ml Saline PBS pH 7 4 containing 0 01 Tween 20 and 0 09 sodium azide RiboMinus Human Mouse Probe in ultrapure water 100 pmol l Hybridization Buffer B5 RiboMinus The components included in the RiboMinus Concentration Concentration Module are listed below Module Store the module at room temperature Binding Buffer L3 Wash Buffer W5 Sterile RNase free Water Spin Cartridges with Collection Tubes Wash Tubes 2 0 ml
12. below consists of a ribonucleoside linked between the 2 oxygen and 4 carbon atom of the methylene ring Braasch and Corey 2001 LNA RNA This configuration locks the sugar backbone resulting in an increase in Tm melting temperature Incorporation of 5 7 LNA monomers into an oligonucleotide does not affect the ability of the oligonucleotide to bind DNA or RNA but increases the stability of the oligonucleotide RNA complex McTigue et al 2004 Oligonucleotides containing LNA are used in hybridization assays requiring high specificity and reproducibility The RiboMinus Magnetic Beads are streptavidin coated magnetic beads used for the removal of probe rRNA complexes from the sample The beads bind to the biotin labeled probe complexed with rRNA or the probe alone The beads are 1 um polystyrene beads with a magnetic core that is strong enough to separate the bound complex from the solvent in a short period of time page 4 for specifications The beads do not promote non specific binding of any other RNA molecules The size and the biotin binding capacity of the RiboMinus Magnetic Beads is optimized for use with this kit and results in gt 95 depletion of rRNA using 10 ug total RNA as the starting material Avoid using any other streptavidin coated magnetic beads with this kit Continued on next page Selective Hybridization and Removal of rRNA Continued Experimental The figure below depicts the expe
13. invitrogen RiboMinus Transcriptome Isolation Kit For efficient isolation of RiboMinus RNA Catalog no K1550 01 Version B 3 January 2005 25 0966 ii Table of Contents Table OF Comers a aa oil s N OE EE EAE AEE iii Experienced Users Procedure ccccccccecceceecceeeeeteeeeeeeeeeeess iv Kit Contents and Storage ss eN a See ge ede OS ee Re N Ge Vi Accessory Prodiels Re ER DEE Ede EN Ge Ge EN Ge ee eN ee Ee viii ei EE EE HO N 1 Product Specifications eie ee ee Ee ee Ee RE EE ee ee ee ee Ee EE 4 Experimental Overvi W eise esse ee ee ee ee ee eke ee Re Re AA AA eke AA Re AA Re AA Re ee 5 Preparing Total RNA RS KEER ERGE ER EE se Ge OR Gee EN Ee DEE SERE Rg EE served 6 Selective Hybridization and Removal of rRNA i ee ee ee 7 Concentrating RiboMinus RNA Fraction 13 Analyzing RiboMinus RNA ee ee ee ee ee ee ee ee ee 17 Troubleshooling ss ER a a ee DEE 19 Technical SOIC cs ED aiaa DR Ee ss 21 References RE TE EO ase 22 iii Experienced Users Procedure Introduction This quick reference sheet is included for experienced users of the RiboMinus Transcriptome Isolation Kit If you are a first time user follow the detailed protocol in this manual Isolating Total You will need to isolate high quality total RNA from cells or RNA tissues using a method of choice prior to using this kit You will need 2 10 ug total RNA per reaction Selective Perform hybridization of your total R
14. l RNA transcripts of interest enabling whole transcriptome analysis The isolated RiboMinus RNA is suitable for use in downstream applications such as microarray analysis qRT PCR and cDNA library construction Continued on next page Overview Continued Advantages Using the RiboMinus Human Mouse Transcriptome Isolation Kit to isolate RiboMinus RNA rRNA depleted RNA provides the following advantages e Rapid and efficient isolation of high quality RiboMinus RNA using probes specific to 18S and 28S human and mouse rRNA and spin column based centrifugation e Specifically designed to isolate RiboMinus RNA enriched in polyadenylated polyA mRNA non polyadenylated RNA pre processed RNA tRNA and small rRNAs 5S rRNA 5 85 rRNA e Minimal contamination from rRNA molecules and genomic DNA e Reliable performance of the RiboMinus RNA in downstream applications such as microarray analysis cDNA library construction and qRT PCR Product Specifications System Specifications RiboMinus Probe Specifications RiboMinus Magnetic Bead Specifications RiboMinus Concentration Module Specifications Starting Material rRNA Removal RiboMinus RNA Yield Probe Contents Probe Specificity Probe Size Probe Label LNA Content Probe Mixture Concentration 2 10 ug total RNA gt 95 1 pg from 10 ug total RNA 2 probes each for 185 and 28S rRNA Human and mouse 18 19 oligon
15. l is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service please contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to the specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whethe
16. nce from rRNA that account for 90 95 RNA species in total RNA The RiboMinus Human Mouse Transcriptome Isolation Kit is based on the selective removal of human or mouse abundant large ribosomal RNA molecules 18S and 28S from total RNA and concentrating the RiboMinus RNA enriched fraction Total RNA is hybridized with human and mouse rRNA sequence specific 5 biotin labeled oligonucleotide probes RiboMinus Human Mouse Probe The rRNA 5 biotin labeled probe complex is removed from the sample with streptavidin coated magnetic beads RiboMinus Magnetic Beads The RiboMinus RNA sample is then concentrated using spin column based centrifugation protocol The conditions for binding are adjusted for the RiboMinus RNA sample with ethanol and Binding Buffer L3 The sample is loaded onto a spin cartridge The RiboMinus RNA binds to the silica based membrane in the cartridge and impurities are removed by thorough washing with Wash Buffer The RNA is then eluted in sterile RNase free water TM For details on RiboMinus Human Mouse Probe and RiboMinus Magnetic Beads see page 7 Continued on next page Overview Continued RiboMinus RNA Drawbacks of RNA Purification Methods Downstream Applications The large ribososmal RNA depleted RNA fraction is termed as RiboMinus RNA fraction The RiboMinus RNA fraction contains polyadenylated polyA mRNA non polyadenylated RNA pre processed
17. ng the sample quickly by placing the tube in cold water 5 While the sample is cooling down prepare the magnetic beads as described below Follow the recommendations on page 10 for handling beads and performing the washing steps 1 Resuspend the RiboMinus Magnetic Beads in its bottle by inverting and gently tapping the bottle repeatedly 2 Pipet 500 ul of the bead suspension into a sterile RNase free 1 5 ml microcentrifuge tube Procedure continued on the next page Continued on next page 11 Selective Hybridization and Removal of rRNA Continued Preparing Procedure continued from previous page RiboMinus 3 Magnetic Beads continued 4 5 8 Removing 1 rRNA 2 3 4 5 6 7 8 12 Place the tube with the bead suspension on a magnetic stand for 1 minute The beads will settle to the bottom of the tube Aspirate and discard the supernatant Add 500 ul sterile RNase free water supplied with the kit to the beads and resuspend beads by alternately inverting and gently tapping the tube Place the tube on a magnetic stand for 1 minute Gently aspirate and discard the supernatant Repeat Steps 4 5 once Resuspend beads in 500 ul Hybridization Buffer B5 Place the tube on a magnetic stand for 1 minute Gently aspirate and discard the supernatant Resuspend beads in 200 ul Hybridization Buffer B5 and keep the beads at 37 C until use Set a water bath or heat block to 37 C After
18. omic Perform DNase I digestion on the RNA DNA sample after elution to remove any contamination genomic DNA contamination Inhibition of Presence of ethanol Traces of ethanol from the Wash Buffer downstream in purified RNA W5 can inhibit downstream enzymatic enzymatic sample reactions reactions To remove Wash Buffer W5 discard Wash Buffer flow through from the collection tube Place the spin cartridge into the collection tube and centrifuge the spin cartridge at maximum speed for 2 3 minutes to completely dry the cartridge 20 Technical Service Contact Us For more information or technical assistance please call write fax or email Additional international offices are listed on our web page www invitrogen com Corporate Headquarters European Headquarters Invitrogen Corporation Invitrogen Ltd 1600 Faraday Avenue Inchinnan Business Park Carlsbad CA 92008 USA 3 Fountain Drive Tel 1 760 603 7200 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Tech Fax 44 0 141 814 6117 E mail tech_service invitrogen com E mail eurotech invitrogen com MSDS Requests Limited Warranty To request an MSDS visit our Web site at www invitrogen com On the home page go to Technical Resources select MSDS and follow instructions on the page Invitrogen is committed to providing our customers with high quality goods and services Our goa
19. ple preparation waste as it forms reactive compounds and toxic gases when mixed with bleach or acids The RNA can be eluted from the cartridge using a single 100 ul elution or two 50 ul elution The recovery of RNA is gt 96 with a single 100 pl elution while the recovery of RNA is 81 with the first 50 ul elution and gt 90 with the second 50 ul elution Continued on next page 13 Concentrating RiboMinus RNA Fraction Continued Experimental The figure below depicts the experimental outline for Outline concentrating the RiboMinus RNA using a spin column based centrifugation procedure Add Binding Buffer L3 and 100 ethanol to rRNA depleted sample Load sample onto the spin cartridge Wash cartridge with Wash Buffer W5 twice Elute RiboMinus RNA with water into Recovery Tube lt gt Carl Ca 14 Concentrating RiboMinus RNA Fraction Continued Materials Needed Before Starting Binding Step RiboMinus RNA sample from Step 7 page 12 96 100 ethanol Microcentrifuge capable of centrifuging gt 10 000 x g Components supplied with the kit Binding Buffer L3 Wash Buffer W5 Sterile RNase free Water Spin Cartridge and Collection Tubes Wash Tubes and Recovery Tubes Add 6 ml 96 100 ethanol to 1 5 ml Wash Buffer W5 included with the kit Store the Wash Buffer W5 with ethanol at room temperature To the sample from Step 7 page 12 add 500 ul Binding Buffer L3 and
20. r expressed or implied including any warranty of merchantability or fitness for a particular purpose 21 References Braasch D A and Corey D R 2001 Locked Nucleic Acid LNA Fine tuning the Recognition of DNA and RNA Chem Biol 1 1 7 McTigue P M Peterson R J and Kahn J D 2004 Sequence dependent Thermodynamic Parameters for Locked Nucleic Acid LNA DNA Duplex Formation Biochemistry 43 5388 5405 Ruan Y Le Ber P Ng H and Liu E 2004 Interrogating the Transcriptome Trends Biotechnol 22 23 30 2004 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use RNase AWAY and TRIzol are registered trademarks of Molecular Bio Products Inc LNA is a trademark of Exiqon A S LabChip is a registered trademark of Caliper Life Sciences Inc 22 invitrogen Corporate Headquarters Invi Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech service invitrogen com For country ific contact information visit our web site at www invitrogen com
21. rimental outline for Outline hybridization of rRNA to specific probes and removal of rRNA Isolate total RNA Hybridize total RNA with the RiboMinus Probe 70 75 C 5 min Cool slowly Prepare RiboMinus Beads in Hybridization Buffer B5 Allow rRNA probe complex binding to RiboMinus Beads 37 C 15 min Remove rRNA probe complex using RiboMinus Beads Selective Hybridization and Removal of rRNA Continued Materials You will need the following items Needed e Total RNA see previous page Magna Sep Magnetic Particle Separator page viii or equivalent Sterile RNase free microcentrifuge tubes Water baths or heat blocks Components supplied with the kit RiboMinus Magnetic Beads keep on ice until use RiboMinus Human Mouse Probe keep on ice until use Hybridization Buffer B5 Sterile RNase free Water l 7 Follow the recommendations below for best results During the mixing and washing steps of the magnetic beads mix beads by inverting the tube repeatedly or using a vortex A low speed centrifuge pulse may be required to remove beads stuck in the tube cap Avoid mixing by pipetting up and down as it results in bead loss During all washing steps with beads add water or buffer to the tube containing beads while the tube is still on a magnetic stand to prevent drying of beads Remove the tube from the magnet and resuspend the beads as described above Do not allow the beads to dry
22. rm incubation for 1 minute with water before centrifugation RNA quantitation Be sure the RNA quantitation using UV performed with absorbance is performed with 10 mM water Tris HCl pH 7 0 page 17 to accurately measure the UV absorbance Incomplete Too much total RNA The protocols in this manual are removal of used designed to purify RiboMinus RNA rRNA from 10 ug total RNA If you are using more than 10 pg total RNA be sure to adjust the reagent volumes accordingly and use sufficient probe and beads to ensure gt 95 removal of rRNA this may need some optimization Low amount of magnetic beads or probe used Be sure to use the recommended amounts of RiboMinus Probe and RiboMinus Magnetic Beads for efficient removal of rRNA Continued on next page 19 Troubleshooting Continued Problem Cause Solution Incomplete Improper handling To obtain the best results with removal of or drying of beads RiboMinus Magnetic Beads follow the rRNA guidelines on page 10 for washing and mixing the beads and aspirating the supernatant Do not allow the beads to dry as drying reduces the bead efficiency RNA RNA contaminated Follow the guidelines on page 6 to degraded with RNase prevent RNase contamination Poor quality starting Always use fresh samples or samples materials frozen at 80 C for isolation of total RNA Be sure to check the quality of your total RNA prior to use Gen
23. se and 18S rRNA band 1 9 kb e 28S band should be twice the intensity of the 18S band Selective Hybridization and Removal of rRNA Introduction RiboMinus Human Mouse Probe Instructions are provided in this section for selective hybridization of rRNA to the RiboMinus Probe and removal of rRNA using RiboMinus Magnetic Beads See page 9 for an experimental outline The RiboMinus Human Mouse Probe is an oligonucleotide probe mixture containing 2 probes each specific for 18S rRNA and 285 rRNA page 4 for specifications The probe is designed to hybridize with highly conserved regions of the human 18S and 28S rRNA The probe also contains sufficient homology to hybridize efficiently against mouse rRNA Each probe is single stranded and contains 5 7 LNA Locked Nucleic Acid monomers incorporated at specific locations The incorporation of LNA see next page for details on LNA into the oligonucleotide probe increases the depletion efficiency of the rRNA from the samples without increasing the amount of beads or probe concentration The 5 end of each probe is conjugated to biotin to allow removal of rRNA probe complexes by binding to streptavidin RiboMinus Magnetic Beads see next page Continued on next page Selective Hybridization and Removal of rRNA Continued LNA Locked Nucleic Acid RiboMinus Magnetic Beads The structure of the LNA Locked Nucleic Acid monomer see figure
24. ta is used to analyze 18S 28S peak ratio RNA degradation and RNA concentration RiboMinus RNA was purified using 10 ug total RNA from 293F cells as described in this manual Samples 5 ul eluate were analyzed on a 0 8 E Gel agarose gel and imaged to visualize RNA Lanel 1 ul1Kb Plus DNA Ladder Lane 2 Control sample 1 purification procedure performed in the absence of RiboMinus Beads and Probe Lane 3 Control sample 2 purification procedure performed in the absence of RiboMinus Probe only Lanes 4 6 Purified RiboMinus RNA samples Lanes 4 6 show efficient removal of 18S and 28S rRNA bands from the purified RiboMinus RNA samples and Lane 3 shows absence of any non specific rRNA removal Troubleshooting Introduction Review the table below to troubleshoot problems that you may encounter using the RiboMinus Human Mouse Transcriptome Isolation Kit Problem Cause Solution Low RNA Low RNA content Various tissues have different RNA yield content and the yield is dependent on the sample Incorrect binding For efficient binding of RiboMinus conditions RNA to the spin column always add 500 ul Binding Buffer L3 and 600 pl 100 ethanol to the sample prior to loading onto the spin cartridge Ethanol not added to Be sure to add 96 100 ethanol to Wash Wash Buffer W5 Buffer W5 as described on page 15 Incorrect elution Add water to the center of the column conditions and perfo
25. ucleotides 5 biotin label Each probe contains 5 7 LNA monomers in the oligonucleotide 100 pmol pl For details on the probe see page 7 The RiboMinus Magnetic Beads are streptavidin coated magnetic beads Bead Binding Capacity gt 3400 pmoles free biotin per mg RiboMinus Magnetic Beads Bead Size 1 um diameter Magnet Particle Superparamagnetic polydisperse core shell polystyrene particles Concentration 12 mg ml Specific Gravity 1 1 1 4 g cm For details on the beads see page 8 Binding Capacity Column Reservoir Capacity Wash Tube Capacity Recovery Tube Capacity Centrifuge Compatibility Elution Volume 1 mg nucleic acid 700 pl 2 0 ml 1 7 ml Capable of centrifuging at gt 10 000 x g 50 100 pl Experimental Overview Introduction The flow chart for isolating transcriptome using the RiboMinus Human Mouse Transcriptome Isolation Kit is shown below Isolate total RNA Hybridize total RNA with the RiboMinus Probe 70 75 C 5 min Cool slowly Prepare RiboMinus Beads in Hybridization Buffer B5 Allow rRNA probe complex binding to RiboMinus Beads 37 C 15 min Remove rRNA probe complex using RiboMinus Beads Add Binding Buffer L3 and ethanol to the supernatant Load sample onto the spin cartridge Wash the cartridge twice with Wash Buffer W5 Elute RNA in RNase free water Preparing Total RNA Introduction General Handling of RN
26. www invitrogen com or contact Technical Service page 21 Product Quantity Catalog no RNase AWAY 250 ml 10328 011 UltraPure DEPC treated Water 1L 750023 UltraPure DNase RNase Free Distilled 500 ml 10977 015 Water Quant iT RNA Assay Kit 1000 assays Q 33140 Micro to Midi Total RNA Purification 50 reactions 12183 018 System TRIzol Reagent 100 ml 15596 026 DNase I 20 000 units 18047 019 DNase I Amplification Grade 100 units 18068 015 Magna Sep Magnetic Particle Separator 1 K1585 01 viii Overview Introduction System Overview The RiboMinus Human Mouse Transcriptome Isolation Kit provides a novel and efficient method to isolate RNA molecules of the transcriptome devoid of large ribososmal RNA rRNA from total RNA for transcriptome analysis The purification method is not dependent on the polyadenylation status or presence of a 5 cap structure on the RNA See below for details on the purification protocol The isolation of RNA fraction depleted of ribososmal RNA is achieved by the selective removal of the large 18S and 28S human and mouse rRNA molecules from total RNA The resulting rRNA depleted RNA fraction is termed as TM RiboMinus RNA fraction see next page for details Using the kit to isolate RiboMinus RNA results in efficient 295 removal of large 185 and 28S rRNA molecules from 10 ug total RNA enabling the analysis of the whole transcriptome without any interfere
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