QuickGene RNA tissue kit S (RT-S)
Contents
1. Make sure to use recommended volume of the DNase to have sufficient activity 7 Supplying the precipitates in reagents Cause Possible Solution Stored at low temperature Store solutions at 15 C to 28 C If the precipitates are contained incubate the bottle in a water bath at 37 C and mix with inversion the bottle intermittently until the precipitates are dissolved 8 The collection tubes are empty after the elution Cause Possible Solution Missed the discharge Set the discharge tray and check the collection holder and cartridge holder setting up into correct positions Press the DISCHARGE after closed the front cover of the instrument See the QuickGene series user s manual 15 10 Ordering Information Product Cat QuickGene series Automatic Nucleic Acid Isolation Systems QuickGene DNA tissue kit S DT S Dedicated reagent kit for QuickGene series to isolate the Genomic DNA from the tissue QuickGene DNA whole blood kit S DB S Dedicated reagent kit for QuickGene series to isolate the Genomic DNA from whole blood _QuickGene RNA tissue kit S _ RT S Dedicated reagent kit for QuickGene series to purify the total RNA from the tissue QuickGene RNA cultured cell kit S RC S Dedicated reagent kit for QuickGene series to purify the total RNA from cultured cell QuickGene Plasmid kit S PL S Poa aaa a a calla Trade Mark Fa2. Make sure to add required volume of ethanol to the Wash Buffer WRT prior to use Use of the old Wash Buffer WRT including ethanol Flash remaining Wash Buffer WRT including ethanol which may be one day old or more in the instrument prior to use Lysate not fully applied to Cartridge s CA If aggregates are present in the lysate apply them with the lysate to the cartridge Insufficient amounts of reagents used Make sure that sufficient amount of reagent are in the reagent bottles Insufficient volume of DNase reaction buffer has been added for isolation with DNase treatment Make sure to add specified volume of DNase reaction buffer to DNase solution The membrane is damaged when DNase solution is added for isolation with DNase treatment Avoid physical contact to the membrane when DNase solution is added RNA has been degradated See 4 section 2 Low purity A260 A280 Cause Possible Solution Excess amount of sample was used Reduce the amount of tissue sample to below the specified amount See Table 3 3 Clogging the cartridge Cause Possible Solution Insufficient homogenization following the addition of Lysis Buffer LRT containing 2 ME Allow sufficient homogenization until the sample is uniformly homogeneous immediately after Lysis Buffer LRT addition Excess amount of sample was used Reduce the amount of t
3. If you do not use the eluted total RNA for any experiments immediately after covering the Caps CAP on the Collection Tubes CT tightly store at 20 C or 80 C 11 12 8 3 total RNA isolation using the QuickGene series Automatic Nucleic Acid Isolation System Notice System set up and basic operations Please read the user s manual of QuickGene series Automatic Nucleic Acid Isolation System circumstantially for the details before using the system 1 Selection of isolation mode Select RNA TISSUE or RNA TISSUE PLUS mode for total RNA isolation from tissue with the kit 2 Setting of cartridges and tubes Open the front cover of the instrument and set the collection and waste tubes in the collection tube holder e Use the specified Collection Tubes CT and Waste Tubes WT including in the kit Attach the cartridge holder to the instrument and set 1 8 cartridges in the cartridge holder e Use the specified Cartridges CA Notice Refer to the user s manual for the QuickGene series Automatic Nucleic Acid Isolation System for details of setting cartridges and tubes Incorrect cartridge placement may result in the solution spilling or improper isolation Wear gloves during the experiments to avoid nuclease contamination 3 Setting of reagents Prepare the required volume see 8 1 Preparation of reagents of Wash Buffer WRT with gt 99 ethanol and Elution Buffer CRT into the tubes set them to the holder and put t
4. of elution volume less than default volume minimum 50 ul In case of setting to 50 ul yield may decline If you do not use the eluted total RNA for any experiments immediately after covering the Caps CAP on the Collection Tubes CT tightly store at 20 C or 80 C 10 lt Preparation workflow RNA TISSUE PLUS mode with DNase treatment gt Homogenizer tube Add slice of tissue 5 mg lt Add LRT with 2 ME 350 u Homogenize ex Rotor stator homogenizer Homogenization Pestle for micro centrifuge tube or Beads mill homogenizer Centrifuge 28 000xg 3 min Room temperature Transfer 350 of supernatant to new 1 5 ml micro tube Add SRT 175 Mix thoroughly by vortexing for 15 sec Flash spin down lt Add gt 99 Ethanol 175 ul Mix thoroughly by vortexing for 1 min Flash spin down Lysate Transfer the whole lysate to the cartridge of QuickGene series Automatic Nucleic Acid Isolation System Select RNA TISSUE PLUS mode Press START button After the first washing step display shows START SW gt RESTART Add DNase solution manually 40 ul Press START button Y total RNA Default elution volume 100 4l Details 1 5 Refer to lt Preparation workflow RNA TISSUE mode without DNase treatment gt 6 Add the recommended DNase manually after the first washing step display shows START SW
5. u Homogenize ex Rotor stator homogenizer Homogenization Pestle for micro centrifuge tube or Beads mill homogenizer 2 Centrifuge 28 000xg 3 min Room temperature Transfer 350 of supernatant to new 1 5 ml micro tube i l Add SRT 175 3 Mix thoroughly by vortexing for 15 sec Flash spin down fi Add gt 99 Ethanol 175 ul 4 Mix thoroughly by vortexing for 1 min Flash spin down Lysate Transfer the whole lysate to the cartridge of 5 QuickGene series Automatic Nucleic Acid Isolation System Select RNA TISSUE mode L Press START button 6 total RNA Default elution volume 100 Details The membrane will be clogged when excessive amounts of tissue sample applied to cartridge Use LRT with added 2 ME Please add 2 ME 10 1 1 ml LRT to LRT before using When using the frozen sample keep them frozen just before homogenize Recommended homogenizers are follows three Please read the user s manual of homogenizer before the operation The maximum sample volume using the Homogenization Pestle for micro centrifuge tube may become smaller than using rotor stator homogenizer or beads mill homogenizer a Rotor stator homogenizer Put tissue sample and 350 u1 of LRT with 2 ME to a fresh micro tube and immediately homogenize with Rotor stator homogenizer until the sample is uniformly homogeneous Please optimize the rotation speed and time for
6. W FUJIFILM HANDBOOK QuickGene RNA tissue kit S RT S For Isolation of total RNA from tissue samples Contents Introduction Components of the kit Storage conditions Other required materials not supplied in this kit Safety warnings Precautions Quality controls Protocols 8 1 Preparation of reagents 8 2 Sample preparations 8 3 total RNA isolation using the QuickGene series Automatic Nucleic Acid Isolation System Troubleshooting Ordering Information Contact Information Appendix 1 1 2 3 4 5 6 7 8 Warning For research use only Not recommended and intended for diagnostic or clinical application for human and animals 1 Introduction Fuji Photo Film Co LTD developed and patented an evolutionary porous membrane to immobilize nucleic acid Because of its large specific surface area and uniform amp fine porousness QuickGene successfully isolates total RNA with high yield moreover with its patented thin membrane it eliminates most contaminants QuickGene also uses pressured filtration technology which cannot be successfully utilized with typical glass membranes by using pressured filtration technology new compact and automatic instruments for rapid nucleic acid purification can be produced successfully When QuickGene RNA tissue kit S is used with the QuickGene series Automatic Nucleic Acid Isolation System high quality and high yield total RNA can be isolated and also puri
7. f accidental ingestion In case of contact between the reagents and eyes skin or clothing wash immediately with water Wear laboratory coat gloves and safety glasses during experiments Solubilization Buffer SRT Don t put reagents in eyes and be careful of accidental ingestion In case of contact between the reagents and eyes skin or clothing wash immediately with water Wash Buffer WRT Don t put reagents in eyes and be careful of accidental ingestion In case of contact between the reagents and eyes skin or clothing wash immediately with water Elution Buffer CRT Don t put reagents in eyes and be careful of accidental ingestion In case of contact between the reagents and eyes skin or clothing wash immediately with water e Keep away the Lysis Buffer LRT from heat Do not mix with disinfectants such as bleach e For disposal of waste fluid and consumables When using potentially infectious samples for experiments dispose them according to applicable regulations 6 Precautions e Refer to the MSDS Material Safety Data Sheet for specific recommendations on properties and handling The MSDS can be obtained from the World Wide Website http lifescience fujifilm com e Refer to the user s manual for the QuickGene series Automatic Nucleic Acid Isolation System before using lt Prevention against RNase contamination gt e Wear the disposable gloves when you have been handling the RNA and or k
8. fied from tissue samples In addition total RNA from 8 sets of tissue lysate samples can be simultaneously extracted in only 13 minutes without using not only spin columns but also hazardous solvent such as phenol The purified high quality total RNA is suitable for RT PCR northern blot analysis and other applications Please read this handbook carefully before using the kit 2 Components of the kit The kit includes the reagents necessary for 96 sets of total RNA isolation Lysis buffer LRT Solubilization buffer SRT Wash buffer WRT Elution buffer CRT Cartridges CA Collection tubes CT Caps CAP Waste tubes WT 3 Storage conditions Store all reagents at 15 C to 28 C 4 Other required materials not supplied in this kit Reagents e gt 99 Ethanol e 2 mercaptoethanol 2 ME e RNase free PBS e DNase For optional process Recommended products are listed as below RQ1 RNase Free DNase Promega Cat No M6101 DNase I Amplification Grade Invitrogen Cat No 18068 015 DNase Amplification Grade Sigma Cat No AMP D1 Deoxyribonuclease RT Grade Nippon Gene Cat No 313 03161 DNase I RNase Free Ambion Cat No 2222 RNase Free DNase Set QIAGEN Cat No 79254 Instruments and equipments e QuickGene series Automatic Nucleic Acid Isolation System e 1 5 ml Micro centrifuge tubes e Centrifuge tubes see Table1 e Micropipettes and t
9. gt RESTART 6 1 Prepare the recommended DNase Product Name Manufacturer Cat No Preparation Final Conc RQ1 RNase Free DNase Promega M6101 DNase Amplification Grade Invitrogen 18068 015 PEFR 1 20 U 40 ul DNase Amplification Grade Sigma AMP D1 Deoxyribonuclease RT Grade Nippon Gene 313 03161 DNase RNase Free Ambion 2222 2 40 U 40 ul RNase Free DNase Set 1 QIAGEN 79254 3 3 4 Kunitz unit 40 4I 1 Dissolve 1 500 Kunits units of DNase with 550 ul of RNase Free water before preparing the DNase reaction solution Preparation 1 1 U UI DNase 204l 10xReaction Buffer 4ul RNase Free water 16ul Preparation 2 2 U U DNase 20ul 10xReaction Buffer 4ul RNase Free water 16ul Preparation 3 2 7 Kunitz unit U DNase 1 25ul Buffer RDD 35 Ul RNase Free water 3 7541 6 2 Addition of DNase Open the front cover of the instrument and add 40 ul of DNase reaction solution to each cartridge by using micropipettes Do not touch the membrane by pipet tip when DNase reaction solution is added Close the front cover and hold for 5 min 6 3 Default waiting holding time of DNase treatment is 5 min You may change the setting of time as the parameter of a program 7 Default elution volume is 100 ul but you may change the setting of elution volume less than default volume minimum 50 ul In case of setting to 50 ul yield may decline
10. he holder to the designated positions of instrument Notice Wear gloves during the handling of reagents to avoid nuclease contamination e Read the user s manual for the QuickGene series Automatic Nucleic Acid Isolation System for details for setting reagents 4 Discharge Set the discharge tray and check the collection holder and cartridge holder setting for the correct positions Press the DISCHARGE after closed the front cover of the instrument Notice Because of air in the tubings incorrect volume of reagents may occur without discharge operation 5 Applying the prepared samples Apply all contents of prepared lysate samples see 8 2 Sample preparations into the each Cartridge CA by using micropipettes any aggregates in the lysate should be transfered into the cartridge 6 Isolation Close the front cover of the instrument Confirm the appropriate mode on the operation panel and press the START button When using the 30 mg of mouse liver elution time may extend until 25 min or the cartridge on QuickGene series may clog In this case reduce the tissue sample volume 7 Collection of total RNA After completing the process each sample result is indicated on the operation panel as follow v Check Completed normally Hyphen Not completed normally _ Underscore No cartridge or no sample Open the front cover and remove the Collection Tube s CT from the collection tube holder e As total RNA is e
11. ips e Homogenizer a Rotor stator homogenizer b Homogenization Pestle for micro centrifuge tube c Beads mill homogenizer e Vortex mixer e Micro centrifuge e Tube stands Table1 Recommended centrifuge tubes Size of QuickGene series centrifuge tube holder prose arpa Examples Type of centrifuge tube Large centrifuge tube for WRT BD Falcon 50 ml conical tube Standard Small centrifuge tube for CRT BD Falcon 15 ml conical tube r BD Falcon 175 ml conical tube Large Large centrifuge tube for WRT BD Falcon 225 ml conical tube Small centrifuge tube for CRT BD Falcon 50 ml conical tube Centrifuge tubes are used with the QuickGene series Automatic Nucleic Acid Isolation System as containers for the Wash buffer WRT with ethanol and Elution buffer CRT 5 Safety warnings Warning For research use only Not recommended and intended for diagnostic or clinical application for human and animals e All reagents and items should be considered chemically and biologically hazardous Wearing a laboratory coat gloves and safety glasses during the experiments are highly recommended In case of contact between the reagents and the eyes skin or clothing wash immediately with water See the Material Safety Data Sheet for specific recommendations http lifescience fujifilm com Lysis Buffer LRT Poisonous if swallowed Don t put reagents in eyes and be careful o
12. issue sample to below the specified amount See Table 3 4 RNA degradation Cause Possible Solution Improper tissue sample storage conditions Quickly freeze the tissue in liquid nitrogen that will not be used immediately then store them at 80 C or below Avoid use of tissue samples kept at room temperature for a long time and or thawing during handling RNase contamination Although all buffers cartridges and collection tubes are supplied RNase free RNase contamination may occur during preparation and storage Extreme caution to avoid RNase contamination RNase contamination in DNase for isolation with DNase treatment Use a recommended RNase free DNase Heating of RNA RNA would be degraded when heated Store RNA samples on ice during experiments 5 Subsequent experiments e g RT PCR unsuccessful Cause Possible Solution Improper amount of RNA was used Determine the RNA concentration based on the absorbance at 260 nm Contamination of genomic DNA Isolate the total RNA by DNase treatment RNA PLUS mode RNA has been degradated See 4 section 6 Incomplete of DNA degradation lt RNA PLUS mode gt Cause Possible Solution Membrane not completely soaked in DNase solution Make sure that DNase is evenly distributed over the membrane in the Cartridge s when DNase solution is applied Insufficient DNase activity
13. it because of prevention of RNase contamination e Use the disposable sterilization plastic materials during the operations These materials are almost experience RNase free but not guarantee therefore usually you may not need RNase free process e In case of using the glass or metal materials you have to hot air sterilize them at 200 C more than 16 hr 7 Quality controls e The stability of the reagents is guaranteed for 9 months after purchase if stored at the specified temperature 15 C to 28 C e As part of the stringent of quality assurance program in Fuji Photo Film Co LTD the performance of QuickGene RNA tissue kit S is evaluated routinely on a lot to lot uniformity e QuickGene RNA tissue kit S is tested for contaminations of RNase e Quality and yield of isolated total RNA are checked by measuring the absorbance at 260 nm ratio of absorbance 260 nm 280 nm and RT PCR amplification 8 Protocols 8 1 Preparation of reagents Lysis Buffer LRT Mix thoroughly before using If the precipitates are contained in Lysis Buffer incubate the bottle in a water bath at 37 C and mix with inversion the bottle intermittently until the precipitates are dissolved After dissolving the Lysis Buffer cool down the bottle to room temperature before using Dispense the requirement volume and add 2 ME for 10 ul 1 ml LRT each time Solubilization Buffer SRT Mix thoroughly before using If the precipitates are contained in S
14. lconTM Becton Dickinson and Company The Polymerase Chain reaction PCR is covered by patents owned by Roche Molecular Systems and F 16 Hoffmann La Roche Ltd 11 Contact Information http ifescience fujifilm com Fuji Photo Film Co Ltd LIFE SCIENCE PHOTO IMAGING amp INFORMATION PRODUCTS DIVISION 26 30 Nishiazabu 2 Chome Minato ku TOKYO 106 8620 JAPAN Tel 81 3 3406 2201 Fax 81 3 3406 2158 E mail sginfo tokyo fujifilm co jp Subsidiaries lt United States Canada Mexico gt Fujifilm Medical System U S A Inc 419 West Avenue Stamford CT 06902 U S A Tel 1 203 324 2000 ext 6112 1 800 431 1850 ext 6112 in the U S Fax 1 203 351 4713 E mail SSG fujimed com URL http lifescience fujifilm com lt Europe excl UK and Ireland gt Fuji Photo Film Europe GmbH Heesenstr 31 40549 Dusseldorf Germany Tel 49 211 5089 174 Fax 49 21 1 5089 139 E mail lifescience fujifilmeurope de URL http www fujifilm de lt UK Ireland gt Fuji Photo Film U K Unit 12 St Martinsfs way St Martin s Business centre Bedford MK42 QLF UK Tel 44 1234 245291 Fax 44 1234 245293 E mail lifesciences fuji co uk URL http lifescience fujifilm com lt China gt Fuji Photo Film China Investment Co Ltd 31st floor Hong Kong New World Tower No 300 Huai Hai Zhong Road P R China Tel 86 21 3302 4655 Fax 86 21 6384 3322 E mail wgxiang fujifilm com cn URL http www f
15. luted from the Cartridge s CA using 100 ul of Elution Buffer CRT the volume of recovered total RNA solution will be 100 ul Cover with the Caps CAP on the Collection Tubes CT containing the isolated total RNA tightly 8 Clean up Remove the Waste Tubes WT and dispose the waste fluid according to applicable regulations Remove the cartridge holder and dispose the Cartridges CA Warning Disposal of waste fluid and consumables When using the potentially infectious samples for experiments dispose them according to applicable regulations 13 14 9 Troubleshooting Review the information below to troubleshoot the experiments with QuickGene RNA tissue kit S For system related problems e g when an error message appears see the QuickGene series user s manual 1 Low yield or no RNA obtained Cause Possible Solution Improper storage condition of sample Optimize storage conditions in different sample volume and storage period Avoid use of tissue samples kept at room temperature for a long time and or thawing during handling Insufficient amount of sample was used Increase the amount of tissue sample See Table 3 Insufficient homogenization following the addition of Lysis Buffer LRT containing 2 ME Allow sufficient homogenization until the sample is uniformly homogeneous immediately after Lysis Buffer LRT addition Required volume of ethanol was added to Wash Buffer WRT
16. mg See Table 3 Table 3 Average yield and maximum sample amount of starting material for mouse tissue Mouse Tissue Average yield sample amount Maximum sample amount Liver 20 Ug 5 mg 30 mg Brain 12 ug 30 mg 50 mg Spleen 15 Ug 10 mg 30 mg Kidney 15 Ug 10 mg 20 mg Lung 12 Ug 20 mg 30 mg Heart 10 ug 15 mg 20 mg Thymus 15 Ug 10 mg 20 mg Completely homogenize with using the beads mill homogenizer The yield depends on the sample type and the storage condition These yield are typically data of mouse tissues e Tissue type tissue storage condition and homogenize method may change the maximum sample amount and extend the processing time If the cartridges are clogged reduce the amount of sample e Soak the 5 mg of sliced tissue in the Lysis Buffer LRT and homogenize immediately after sample collection If you do not prepare the samples immediately freeze the tissue with liquid nitrogen and store at 80 C e Keeping the samples at room temperature for a long time and or thawing tissue during handling may degrade RNA or lower the yield e Use calibrated pipets for the buffer dispensing The volumes are adjusted for the best performance of the system e Take all of operation rapidly at room temperature 15 30 C lt Preparation workflow RNA TISSUE mode without DNase treatment gt Homogenizer tube Add slice of tissue 5 mg lt Add LRT with 2 ME 350
17. olubilization Buffer incubate the bottle in a water bath at 37 C and mix with inversion the bottle intermittently until the precipitates are dissolved After dissolving the Solubilization Buffer cool down the bottle to room temperature before using Wash Buffer WRT Provide the concentrated solution Add 40 ml of gt 99 ethanol into the bottle and mix with inversion the bottle gently at the beginning of use Requirements of Wash Buffer WRT with gt 99 ethanol and Elution Buffer CRT Prepare the requirements of Wash Buffer WRT with gt 99 ethanol and Elution Buffer CRT according to the number of samples for isolation refer to the following table Take some of the buffers into each tube and set the tubes in the QuickGene series system tube holder See the user s manual of QuickGene series Automatic Nucleic Acid Isolation System Table 2 Buffer volume and the number of samples to set in the QuickGene System Number of samples WRT with 99 Ethanol CRT 8 26 ml 9 ml 16 44 ml 11 ml 24 62 ml 13 ml 32 80 ml 15 ml 40 99 ml 17 ml 48 117 ml 19 ml 56 135 ml 21 mi 64 154 ml 22 ml 72 172 ml 24 ml 80 190 ml 26 ml 88 209 ml 28 ml 96 227 ml 30 ml 8 2 Sample preparations e Basically the QuickGene RNA tissue kit S is designed for total RNA isolation from 5 mg of mammalian tissue sample However several tissue samples would be able to isolate the total RNA from more than 5
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19. your sample b Homogenization Pestle for micro centrifuge tube Use the exclusive motor and place the Pesile to the motor Put tissue sample and 200 ul of LRT with 2 ME to a fresh micro tube and immediately homogenize with Pestle until the sample is uniformly homogeneous Add 150 ul of LRT in homogenate and mix thoroughly by vortexing with maximum speed for 15 sec Flash spin down the homogenate c Beads mill homogenizer Put tissue sample and 3501 of LRT with 2 ME to a fresh micro tube and immediately homogenize with Beads mill homogenizer until the sample is uniformly homogenous 2 Centrifuge 28 000xg 3 min at room temperature to remove insoluble materials and transfer the 350 ul of supernatant to a fresh micro tube It may be necessary to increasing the rotation speed and time in order to spin down insoluble materials completely 3 Add 175 u of SRT and mix thoroughly by vortexing with maximum speed for 15 sec and flash spin down 4 Add 175 ul of gt 99 Ethanol and mix thoroughly by vortexing with maximum speed for 1 min and flash spin down the lysate completely Incomplete vortexing may cause low yield 5 Transfer the whole lysate to the cartridge of QuickGene series Automatic Nucleic Acid Isolation System and immediately press the start button If any aggregates are present in the lysate in step 4 apply all of them with the lysate to the cartridge 6 Default elution volume is 100 ul but you may change the setting
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