InviMag Virus RNA Mini Kit/ IG User manual
Contents
1. eluted RNA is brownish colored Probable cause pipetting of PKC failed samples transfer failed incomplete reagent buffer transfer failed incomplete blood components settled no too much ethanol added to Wash Buffers incorrect storage of starting material old material combination of reagents from different kits missing required reagent Residual magnetic particles are left in eluate Comments and suggestions ensure that the lyophilized PKC is lyophilized with the appropriate volume of water before usage the sample tube must contain at least 400 ul sample ensure that the supplied Wash Buffers Binding Solution is filled up properly with either ethanol or isopropanol do not reuse bottles more often than described in Tab 1 because they will be rejected by the system in case of large sample volumes gt gt 1 ml carefully premix the sample tube before inserting it into the sample rack ensure that the Wash Buffers have been filled up properly with ethanol as indicated in Tab 1 ensure that the storage of starting material is correct avoid multiple freezing and thawing cycles of the material ensure that the starting material is fresh or stored at appropriate conditions for long time storage at 20 C avoid multiple thawing and freezing cycles of the material old material may contain degraded DNA make sure that only and all reagents belonging to one kit type are used a c2. specifications Note If beads are visible in the eluate transfer the eluate to a new reaction tube and centrifuge for 1 min at maximum speed e g 13000 rpm Different amplification systems vary in efficiency depending on the total amount of nucleic acid present in the reaction Eluates from this kit contain both viral RNA and Carrier RNA whereas the amount of Carrier RNA will greatly exceed the amount of viral nucleic acids Yields of viral RNA isolated from biological samples are normally less concentrated than 1 ug and therefore impossible to determine photometrically Keep in mind that the added Carrier RNA 5 ug per 200 ul sample will account for most of the present RNA The kit is suitable for downstream analysis with NAT techniques for examples qPCR RT qPCR LAMP LCR Diagnostic assays should be performed accordingly to the manufacturer s instructions Quantitative RT PCR is recommended for determination of viral RNA yield In Gel Electrophoresis and in Capillary Electrophoresis RNA extracted with the provided kit looks like degraded cause the kit contains Carrier RNA this is poly A RNA in fragments of 100 up to 1000 bases The kit is not dedicated for applications using this kind of analysis The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 9 InviMag Virus RNA Mini Kit IG 0515 Important notes Important points before starting a protoc
3. In addition cryoprecipitates formed during freeze thawing can give problems If cryoprecipitates are visible they should be centrifuged at app 6 800 x g for 3 minutes The cleared supernatant should be aspirated without disturbing the pellet and be processed immediately This step will not reduce viral titers Stool Best results are obtained with fresh material The collected fresh stool sample can be stored at ambient temperature for at least 1 2 hours at RT but the high content of DNases and RNases can lead to a quick digestion and degradation of the viral RNA The sample should be quickly prepared and processed on the InviGenius Alternatively it can be stored frozen at 80 C for weeks or months Swabs The protocol works with fresh prepared swabs as well as with dried swabs The protocol has not been validated for isolation of RNA from swabs which are stored in special storage buffers of other providers STRATEC Molecular will not take responsibility if other sample types than described above are used or if the sample preparation advices are modified Principle and procedure The InviMag Virus RNA Mini Kit IG procedure comprises following steps after loading of the samples and buffers and starting the automated process Lysis of the virus particles Binding of the viral RNA to the magnetic beads Washing and evaporation of ethanol Elution of viral RNA O O O After lysis the viral RNA binds to the magnetic beads w
4. lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of InviMag Virus RNA Mini Kit IG or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor 4 InviMag Virus RNA Mini Kit IG 0515 Intended use The InviMag Virus RNA Mini Kit IG is designed for fully automated extraction and purification of viral RNA from up to 200 ul serum or plasma based samples Up to 12 samples can be processed using a patented magnetic beads system and the InviGenius robotic platform It is advised to provide at least 550 ul sample per tube dead volume to prevent pipetting distribution errors due to the liquid level detection LLD process The final processed sample volume is 200 ul The nucleic acid isolation protocol is suitable for routinely walk away automated preparation of viral DNA from fresh or frozen sample For reproducible and high yields an appropriate sample storage is essential see Sampling and storage of the starting material page 9 Common collection tubes not provided and anticoagulants EDTA and citrate but not heparin can be used to assemble a set of sample
5. RNA Mini Kit IG except PKC Tube and MAP Solution B should be stored at room temperature and are stable for at least 12 months PKC Tube Lyophilized PKC Tube should be stored at 2 8 C If dissolved in RNase free water storage at 20 C is recommended MAP B Solution The magnetic beads should be stored at 4 C Wash Buffers Wash Buffers charged with ethanol should be stored at room temperature and should be appropriately sealed If any precipitates are visible within the provided solutions solve them by carefully warming up to 30 C Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviMag Virus RNA Mini Kit IG for applications as described in this manual Purchaser must determine the suitability of the product for its particular use Should any product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot the product will be replaced free of charge STRATEC Molecular reserves the right to change alter or modify any product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the InviMag Virus RNA Mini Kit IG have been tested separately against predetermined specifications routinely on
6. The plate can be reloaded by pressing on the offered plate in C For a successful run the InviGenius needs one free lane in the incubator position seven free lanes in the working position and one free lane in the eluate position Please make sure that the depicted lanes on the monitor are consistent with the real lanes in the corresponding positions To avoid contaminations we strongly recommend to not wash reuse disposed plates 21 InviMag Virus RNA Mini Kit IG 0515 Waste management Please make sure that the waste tray is capacity is sufficient for your planned assay If not empty the solid waste Waste management Waste capacity 190 Dropshaft status In place Fill level 0 disposables 0 Number of wasted disposable sheaths 0 Number of wasted disposable tips 0 A Batch definition loading Status Ready ma User Service Figure 11 Waste management screen If you have cleaned the waste tray please use the Empty solid waste button A Batch definition Please select the appropriate assay and check the samples you want to process in this run Batch definition Assay descriptions IA suitable to loaded reagents RVIR_E100_S200 Samples SAMPLE 1 Remove irom batch Version 1 Aliquots Volume 200 200 Waste Batch checking management o_u Status Ready User service Figure 12 Batch definition screen Please select the desire
7. for the InviGenius system 14 General overview of the InviGenius System 15 Preparing and loading of the InviGenius system 16 After the run 24 Maintenance UV sterilization 24 Appendix 26 Example data 26 General notes on handling RNA 27 Troubleshooting 28 Ordering information 29 2 InviMag Virus RNA Mini Kit IG 0515 Kit contents of InviMag Virus RNA Mini Kit IG Store the MAP Solution B at 4 C Store lyophilized Proteinase K Carrier RNA PKC Tube at 2 8 C Store dissolved Proteinase K Carrier RNA at 20 C Store all other kit components at room temperature RT 8 x 12 extractions reagents sufficient for PKC Tube for 8x 800 i working solution Binding Solution empty bottle erie fill with 99 7 lsopropanol final volume 60 ml 60 ml 2x25 ml J Add 60 ml of 99 7 Isopropanol molecular biologic grade into the empty bottle labelled Binding Solution Initial steps Add 60 ml of 96 100 ethanol to the bottle Wash Buffer R1 Add 100 ml of 96 100 ethanol to each bottle Wash Buffer R2 Mix thoroughly and always keep the bottles firmly closed Add 800 ul of the provided RNase free Water to each PKC vial and mix Only prepare as much vials as required for a run 3 InviMag Virus RNA Mini Kit IG 0515 Symbols LOT Lot number Catalogue number Expiry date Temperature limitation a Consult operating instructions D Do not reuse All buffers and kit contents of the InviMag Virus
8. of the provided RNase DNase free water mix thoroughly and store diluted and unused PKC at 20 C Avoid repeated freezing and thawing cycles which will degrade the Carrier RNA and reduce the functionality of the Kit Add 60 ml of 96 100 ethanol to the bottle Wash Buffer R1 Mix thoroughly and always keep the unused bottle firmly closed Add 100 ml of 96 100 ethanol to each bottle Wash Buffer R2 Mix thoroughly and always keep unused bottles firmly closed Internal Extraction Control please check the instruction on page 14 Reagents and equipment to be supplied by user o Measuring cylinder 250 ml o ddH O o Pipette tips o Vortexer o Disposable gloves o 96 100 ethanol o PBS buffer o sopropanol The InviMag Virus RNA Mini Kit IG is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for lsopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol fur die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 10 InviMag Virus RNA Mini Kit IG 0515 Possible primary tubes manufacturer cat no Venosafe 5 5 ml Ref VF O76SDK Terumo Vacuette 2 ml Ref A110500I Greiner bio one Vacuette 9 ml Ref 455036 Greiner bio one BD Vacutainer 2 7 ml Ref 363048 BD Vacutainer 6 ml Ref 367864 BD Vacutainer 10 ml Ref 367525 BD Vacutainer 5 0 ml Re Sarstedt Monovette 8 5 ml PS Tub
9. switched off or used for sample processing We recommend decontaminating the instrument daily 25 InviMag Virus RNA Mini Kit IG 0515 Appendix Example data To show the reproducibility of the viral RNA extraction using the InviGenius system three extractions were performed of an artificial Influenza mastermix with different elution volumes Runfiles RNAVIR 100 RNAVIR_150 and RNAVIR 200 dead volume Soul Real time PCR was performed to quantify the extracted viral RNA 05 O44 Morm Fluor O aa DE 5 10 15 20 25 30 35 40 45 tycke Figure 18 Real time PCR data of the 3 different runs Rep Ct 95 CI RNAVIR_050 a RNAVIR_050 29 2 RNAVIR 050 28 9 RNAVIR 100 RNAVIR 100 7 IRNAVIR 100 RNAVIR 100 RNAVIR 150 RNAVIR 150 RNAVIR 150 RNAVIR 150 Table 1 Ct of three different diluted volumes protocols N 6 Ea EN 9 The InviGenius produced successful and reproducible results in three different runs with different elution volumes 50 ul remains in the plate 26 InviMag Virus RNA Mini Kit IG 0515 General notes on handling RNA RNA is far less stable than DNA It is very sensitive to degradation by endogenous RNases in the biological material and exogenous RNases which are permanently present everywhere in the lab To achieve satisfactory qualitative and quantitative results in RNA preparations contaminations with exogenous RNases has to be reduced as much as possible Avoid handling bacter
10. 2015 04 30 PB618025302501881504 MAP Sol B PB 2015 04 30 PB312113130700611504 Wash B R2 PB 2015 04 30 Unknown Unknown PB218048100000101504 Binding Sol PB 2015 04 30 PB 55020101000351504 Sealing Oil PB 2015 04 30 Status Ready i User service Figure 6 Reagent loading screen of the InviGenius software Insert all provided reagents into the provided reagent rack of the InviGenius system Take care that the bar code labels face to the right side of the loading bay and decap the bottles and tubes The order of the inserted reagents is not crucial because the type and position of a reagent is identified by the unique bar code However the possible loading positions are limited by the size of the used boitles After rack insertion the loading status of the reagents will be shown In case of unsuccessful reagent allocation remove the rack check the bar code orientation and repeat the procedure slowly 18 InviMag Virus RNA Mini Kit IG 0515 Assay Selection Select RVIR E100 S200 assay and proceed with disposable tip loading Assay selection Assay description RVIR_E100_ 200 suitable to loaded No Assay Selected reagents Version 1 Reagent loading Disp tip loading Status Ready mam Ser service Figure 7 Assay selection screen of the InviGenius software Disposable Tip Loading Disposable tips loading Load Pos 1 Tip Load Position 1 Tip 1100 ul filter loading Statu
11. F IN EYES Rinse cautiously with water for several minutes Remove contact lenses if Present and continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 inside of USA 1 800 535 5053 6 InviMag Virus RNA Mini Kit IG 0515 Product characteristics of the InviMag Virus RNA Mini Kit IG The InviMag Virus RNA Mini Kit IG is an ideal tool for efficient and fully automated viral RNA extraction and purification from fresh or frozen samples using magnetic beads in combination with the InviGenius system Starting material Time for preparation 200 ul serum or plasma depends on the sample 70 min for 12 Source and storage samples Note The added Carrier RNA PKC tube will account for 200 ul cell free body fluids 200 ul rinse liquid from swab 200 ul supernatant from cell cultures most of the eluted RNA Quantitative RT PCR is recommended for determination of the viral RNA yield 50 mg stool sample supernatant from stool suspension The RNA isolation process is based on the interaction of nucleic acids with silica coated magnetic particles at adapted buffer conditions The InviGenius instrument will automatically perform all steps of sample and reagent distribution The RNA purification procedure is performed without any user intervention ex
12. Kits in combination with commercially available amplification systems may require introducing an internal control IC into the purification procedure to monitor the efficiency of sample preparation Internal control DNA or RNA IC must be combined with Carrier RNA stock solution or with Carrier RNA Proteinase K stock solution InviMag Universal Kit IG in one mixture For each sample the machine transfers a volume of 40 ul of the stock solution to the lysis mix The vials with PKC Proteinase K and Carrier RNA mixture are dissolved with 800 ul RNase free water Therefore an internal control for 20 samples should be added and added volume must be subtracted from the total volume Example Calculation Per Extraction 4 5 ul of a control is required 4 5 ul RXN X 20 RXN 90 ul PKC stock solution has to be made by adding 800 ul 90 ul 710 ul RNase free water Then 90 ul control DNA is added followed mixing Notes If the indication of amount per reaction is known please calculate by using eluate and template volume If the internal control IC is stable in plasma serum CSF urine respiratory samples whole blood stool transport media or on dried swabs e g armored RNA it can alternatively be added to the sample shortly before beginning sample preparation But consider that a bigger amount of internal control is necessary when using bigger volumes of primary sample tubes If the internal control IC is nake
13. Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1G5j02 IG 05 2015
14. ay parking position The disposable waste tray I is located behind the lower cover of the InviGenius Interaction with the InviGenius instrument is performed by use of the touch LCD J located at the top front right side 15 InviMag Virus RNA Mini Kit IG 0515 Preparing and loading of the InviGenius system Preparing the reagents Before you start dissolve one vial of Proteinase K and Carrier RNA with 800 ul DNAse free water respectively Preparing the system Turn on the InviGenius system using the power switch located on the right back side of the instrument The InviGenius software can be started by double clicking the InviGenius icon located on the desktop Keep the door of the InviGenius system closed during initialization After initialization of the InviGenius system a login screen appears Figure 1 Log in with the provided user name and password Login User identifier er Password ir Status Ready Atm Ser Figure 2 Login screen of the InviGenius software After login the main screen of the InviGenius software is shown Figure 2 Select Loading to start with loading of the system Select Processing to define and run an assay if the system has already been loaded Main Menu management InviGenius Status Ready oa User developer Loading Processing Shutdown Figure 3 Main menu of the InviGenius software 16 InviMag Virus RNA Mini Ki
15. be given implied or expressed The user is responsible to validate the performance of the STRATEC Molecular product for any particular use STRATEC Molecular does not provide validations of performance characteristics of the product with respect to specific applications STRATEC Molecular products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastics are for laboratory use only They must be stored in the laboratory and must not be used for other purposes than intended The product with its contents is not suitable for consumption D InviMag Virus RNA Mini Kit IG 0515 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF forma
16. cept the initial loading of the system thus allowing safe handling of potentially infectious samples Sample cross contamination and reagent cross over is effectively eliminated by the automated purification process The use of unique bar codes for samples and reagents avoids unwanted transpositions The InviGenius instrument uses magnetic rods to transport the RNA binding magnetic particles through the various extraction phases lysis binding washing and elution The volume of buffers and other liquids necessary for RNA isolation is reduced to a minimum Eliminating the direct liquid handling and increasing the automation level results in a fast reliable and robust technique After a sample specific lysis using Lysis Buffer RV and PKC optimal binding conditions are adjusted by addition of Binding Solution The viral RNA binds to the simultaneously added magnetic particles and is separated from the solution by the magnetic rods controlled by the InviGenius system Subsequent to three washing steps of the particle bound nucleic acids using Wash Buffer R1 and Wash Buffer R2 the DNA is finally eluted in Elution Buffer R Due to the high purity the eluted viral RNA is ready to use in a broad panel of downstream applications such as o real time PCR quantitative RT PCR like TaqgMan und LightCycler technologies o or array technologies For the isolation of DNA from a single serum plasma or blood sample STRATEC Molecular offers t
17. d DNA or RNA it is unstable in plasma serum CSF urine respiratory samples whole blood stool transport media or on dried swabs and must not be added directly to the samples Refer to the manufacturer s instructions to determine the optimal amount of internal control IC for specific downstream applications Using an amount other than that recommended may lead to wrong quantification results 14 InviMag Virus RNA Mini Kit IG 0515 General overview of the InviGenius System Figure 1 Frontal view of the InviGenius System There are three plate positions available in the InviGenius system which can be loaded with corresponding plates the incubator k A the working B and the eluate position C Lysis is performed at the incubator position A whereas the washing and elution process is performed at the working position B The eluate containing the extracted nucleic acids will be finally transferred to the eluate position Additionally there are three loading positions available for disposable tip trays D1 D3 and one position E for the disposable sheaths The loading bay F is located at the very right side of the instrument The sample rack is loaded into the far left lane whereas the reagent rack is loaded into the right lanes of the loading bay The Magnetic Separation Head MSH G is located on top of the incubator lid parking position The fully automatic pipettor H is installed above the loading b
18. d assay and recheck the allocated samples that should be processed in this run It is possible to switch between the offered assays by using the two arrow buttons A By default all loaded samples are selected to be processed in this run If samples have to be excluded from the batch exclude them by selecting the corresponding sample and clicking on the Remove from batch button B 22 InviMag Virus RNA Mini Kit IG 0515 Batch checking This screen shows a summary of all checked disposables samples and reagents in one informational screen Please make sure that all required components are loaded correctly In case of any error the problem will be highlighted in by a red font If no errors during the loading steps occurred proceed by pressing the button Batch processing To solve any error click on the red highlighted field and follow the instructions printed on the instrument screen Batch checking Selected assay RVIR_E100S200 01 01 00251 ooogooco0c00000gg eoocoocooooos Sooococooo0o00so eoooocooooooo KOKOKOROKOTOROKOKOROROTO TG vw A 46 i Batch processing User developer eae a Batch definition Status Ready Figure 13 Batch definition screen Batch processing After closing the system door the assay can be started by pressing the Start Button A The door will be locked during the run and the system will start with sample processing The door will only be un
19. e Sarstedt 5ml Ref 55 476 Sarstedt Monovette 4 5 ml Sarstedt Monovette 7 5 ml Sarstedt Monovette 9 0 ml Important indications 1 Minimum volume of samples in primary tubes The procedure of the InviMag Virus RNA Mini Kit IG is optimized for the isolation of viral RNA from up to 200 ul cell free body fluids serum plasma and rinse liquid from swabs as well supernatant from stool suspensions It is advised to provide at least 550 ul sample tube to prevent pipetting distribution errors during processing 2 Sample volume smaller than 200 ul For samples of a smaller volume than 200 ul please fill the sample tube with PBS to a volume of minimal 400 ul 3 Elution volume The final processed sample volume is 200 ul which is eluted in 100u Elution Buffer R contains no EDTA Prevention of cross contamination To comply with the demanding guidelines of in vitro diagnostics we programmed the InviGenius to route the pipettor in such a way that possible contamination risks are minimized However we recommend to apply the supplied well strios and foils beforehand and afterwards on the used wells on the unused wells of the Incubation Plate A and the Working Plate B To prevent any form of salt crystallization of used Lysis Buffer RV or Wash Buffer R1 wells it is recommended to reseal used wells after a run 11 InviMag Virus RNA Mini Kit IG 0515 Scheme of the InviMag Virus RNA Mini Kit IG Add the primary tubes in the
20. e sure that the disposable sheaths are loaded and displayed consistent to the manually loaded sheaths in the rack to ensure correct sheaths pick up Don t remove single disposable sheaths within a row of the sheaths rack if less than 12 samples are processed within one run because there is a sheaths detection sensor installed in the device If less than 12 sheaths picked up by the instrument a warning will be displayed and all picked up sheath will be discarded into the waste before a next row of sheaths will be picked up for testing To avoid contaminations we strongly recommend to not wash reuse any disposed sheaths 20 InviMag Virus RNA Mini Kit IG 0515 Plate Loading Analogous to the previous loading screens the Incubation Plate A Working Plate A and Elution Plate E are loaded within the plate loading screen Figure 9 Microplates Incubator Por Eluate Position Free lanes 8 B i A i i i Disp sheaths Waste P A Back loading management Status Ready i ser service Elution Plate E Figure 10 Plate loading screen In general the Incubation Plate A and Working Plate A identical are used at the incubator and working position whereas at the eluate position the Elution Plate E is used Used plates can be unloaded and reloaded by 1 Pressing the plate position directly A The software will focus at the plate position on the main screen 2 Pressing the Unload button B 3
21. he Invisorb Spin Virus RNA Mini Kit or for 8 96 samples the Invisorb Virus RNA HTS 96 Kits for use on a centrifuge vacuum manifold or other robotic workstations For further information please contact phone 49 0 30 9489 2901 2910 in Germany and from foreign countries phone 49 0 30 9489 2903 2907 or ask your local distributor 7 InviMag Virus RNA Mini Kit IG 0515 Sampling and storage of starting material Best results are obtained using freshly extracted samples As long as the samples are not shock frosted with liquid nitrogen or incubated with RNase inhibitors or denaturing reagents the viral RNA is not secured It is essential that samples are processed as fast as possible on the InviGenius Long term storage at 80 C is recommended Serum and plasma After collection and centrifugation serum and plasma from blood treated with anticoagulants like EDTA or citrate but not with heparin synovial fluid samples or other cell free body fluids swabs as well as stool samples can be stored on ice for 1 2 hours For a short time up to 24 h samples may be stored at 20 C For long term storage we recommend freezing samples in aliquots at 80 C Frozen plasma or serum samples must not be thawed more than once Multiple thawing and freezing before isolating the viral RNA should be avoided It may lead to denaturation and precipitation of proteins resulting in reduced viral titers and therefore reduced yields of viral Nucleic acids
22. hereas contaminations and enzyme inhibitors are efficiently removed during the following three wash steps and highly purified viral RNA is eluted finally This manual contains 3 protocols 8 InviMag Virus RNA Mini Kit IG 0515 Procedure Lysis Samples are lysed at denaturing conditions in an Incubation Plate A at elevated temperatures in the presence of Lysis Buffer RV and a PKC mixture Binding of the viral nucleic acids After adding Binding Solution and MAP Solution B to the lysate the viral RNA is bound to the surface of the magnetic beads Removing residual contaminants Contaminants are efficiently removed using Wash Buffer R1 and R2 respectively while the nucleic acids remain bound to the magnetic beads Elution The viral RNA is finally eluted from the beads using 100 ul Elution Buffer R and transferred to the Elution Plate E The bottom magnets guarantees a magnetic beads free eluate which is ready to use in different downstream applications like real time PCR quantitative RT PCR like TaqMan und LightCycler technologies or array technologies Yield and quality of viral RNA The amount of purified RNA in the InviMag Virus RNA Mini Kit IG procedure depends on the sample type the virus content sample source transport storage and age Yield and quality of isolated viral RNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed accordingly to the manufacturers
23. hould be at least 400 ul to ensure stable processing Please take care that only the first 12 positions of the sample rack can be processed due to the limited number of wells per row of the plastics For correct identification of the sample tubes the unique bar codes must face to the bar code scanner located at the right side of the loading bay After inserting the sample rack in the very left lane of the loading bay an updated screen will show the identifiers read from the sample bar codes Figure 7 In case of unsuccessful sample identification remove the rack check the bar code orientation and reinsert the rack 17 InviMag Virus RNA Mini Kit IG 0515 slowly It is also possible to rename the samples by selecting the corresponding sample by using the arrow fields followed by the Edit button After a certain time about 5 min the bar code scanner is inactivated In that case the user has to restart the scanner with the START SCANNER button if the loading procedure is not finished After successful loading of the samples proceed with reagent loading by selecting Reagent loading on the bottom right hand side of this screen Reagent Loading The reagent loading process is analogous to the sample loading procedure Reagents loading Barcode PB405025130500481504 Elution B R PB 2015 04 30 PB112055130500451504 Lysis B RV PB 2015 04 30 PB 07700230227871504 PKC PB 2015 04 30 PB310108131200361504 Wash B R1 PB
24. ial cultures cell cultures or other biological sources of RNases in the same lab where the RNA purification is to be carried out O Non disposable plastics should be treated before use to ensure that it is RNase free Plastic ware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water You can also take chloroform resistant plastic ware rinsed with chloroform to inactivate RNases All buffers must be prepared from DEPC treated RNase free ddH20 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Change gloves frequently and keep tubes closed Use only sterile disposable polypropylene tubes throughout the procedure these tubes are generally RNase free Keep isolated RNA on ice Do not use kit components from other kits with the kit you are currently using unless the lot numbers are identical To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow for any steps before starting the extraction on the machine This kit should only be used by personnel trained in in vitro diagnostic laboratory practice Storage of RNA Purified RNA can be stored 80 C and is stable for years e g precipitated and stored in 70 ethanol 21 InviMag Virus RNA Mini Kit IG 0515 Troubleshooting Problem pipetting distribution errors low concentration of extracted RNA degraded RNA no assay selectable
25. locked after a run has been successfully finished or if an error occurs that requires user interaction Do not try to force open the door during a run This will cause an abort of the run Batch processing Batch identifier 1005301137439620000025 User interaction Assay description RVIR_E100S200 01 01 00251 request Actual step s Remaining time 00 00 00 Process state Idle Events Timeout waiting for a rack Position reached after several retries Status Ready OTT lt User developer Figure 14 Batch processing screen At the end of the run the viral RNA containing eluate is located in the appropriate eluate position and can be used for further applications 23 InviMag Virus RNA Mini Kit IG 0515 After a run After a run is completed and no additional run shall be started unload all plates and reagents and store them according to GLP guidelines Please keep in mind that the plates could contain infectious material As with all medical clinical and diagnostically equipment all waste liquids tips sheaths and plates should be treated as potentially dangerous bio hazard waste Daily maintenance UV decontamination The InviGenius system is equipped with an internal UV lamp 254 nm wavelength that should be used daily either at the end of the working day or in the morning before a run is started The suggested decontamination time is about 20min To start the UV decontamination go to the main menu
26. n vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks InviGenius InviMag Invisorb Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 InviGenius InviMag and Invisorb are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved 1 InviMag Virus RNA Mini Kit IG 0515 Contents Kit contents of InviMag Virus RNA Mini Kit IG 3 Symbols 4 Storage 4 Quality control 4 Intended use 3 Product use limitation 5 Safety information 6 Product characteristics of the InviMag Virus RNA Mini Kit IG 7 Sampling and storage of starting material 8 Principle and procedure 8 Yield and quality of viral RNA 9 Important notes 10 Preparing reagents and buffers 10 Reagents and equipment to be supplied by user 10 Important indications 11 Prevention of cross contamination 11 Scheme of the InviMag Virus RNA Mini Kit IG 12 Preparing the samples for processing on the InviGenius system 13 Preparing of the internal control
27. of the InviGenius software and select Maintenance Main Menu Loading Data management Maintenance Administration InviGenius Status Ready Sa User developer Processing Shutdown Figure 15 Main screen of the InviGenius software When the sub item Maintenance is opened select UV decontamination 4 UV gt Periodical decontamination maintenance Maintenance procedures x Cleanup Status Ready mn User service Figure 16 Maintenance screen of the InviGenius software 24 InviMag Virus RNA Mini Kit IG 0515 In the UV decontamination menu adjust the exposure time A and finally press the Start button B During the decontamination process the instrument door will be locked to prevent any UV radiation release in the lab Warning UV radiation is harmful It causes serious burns of the skin and leads to irreparable damage of the eyes and skin Ensure that no lab personnel is submitted to direct UV light Do not try to force open the instrument door during the decontamination process UV decontamination A 00 15 00 J HH MM SS Remaining time 00 00 00 Duration UV decontamination status There was no UV decontamination completed N Status Ready memm User service Figure 17 UV decontamination screen When the decontamination is finished go back to the main menu by using the Back button The device is now decontaminated and can be either
28. ol Important points before starting a protocol Immediately upon arrival of the product inspect the kit and its components as well as the package for any apparent visible damages and correct quantities If there are any unconformities please notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 6 Do not use damaged kit components since their use may lead to poor kit performance o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard contaminated gloves immediately Do not combine components of different kits Avoid microbial contaminations of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow o This kit should only be used by trained personnel O O O Preparing reagents and buffers Before starting a run bring all reagents to room temperature Where necessary gently mix and redissolve any precipitates by incubating at 30 C Swirl gently to avoid foaming Lysis Buffer RV MAP Solution B and Elution Buffer R are ready to use 8 x 12 viral RNA extractions Add 60 ml of 99 7 Isopropanol molecular biologic grade into the empty bottle Resuspend lyophilized PKC by addition of 800 ul
29. ombination of reagents belonging to different kit types is not supported centrifuge the eluate plate at full speed for 1 min and transfer supernatant to a new plate tube 28 InviMag Virus RNA Mini Kit IG 0515 Ordering information Product Package size Catalogue No InviMag Virus RNA Mini Kit IG 8 x 12 preparations 2443120100 Related products Invisorb Spin Virus RNA Mini Kit 50 preparations 1040300200 Invisorb Spin Virus RNA Mini Kit 250 preparations 1040300300 Invisorb Virus RNA HTS 96 Kit X 4 x 96 preparations 7143310300 Invisorb Virus RNA HTS 96 Kit X 24 x 96 preparations 7143310400 InviMag Virus RNA Kit KF96 2 X 96 preparations 7443300100 InviMag Virus RNA Kit KF96 5 x 96 preparations 7443300200 InviGenius and consumables InviGenius 1 unit 5011100000 Sheath Box Conductive filter tips 1 ml 2 x 2 rack pack 384 pieces 5 Waste Trays 120 sample tubes Sheath Bundle 10 x 48 pieces 5011100300 Sheaths 1000 pieces 5011100200 Conductive filter tips 1 ml 10 x 96 pieces 5011100400 Waste tray IG 25 pieces 5011100100 Conductive filter tips 1 ml 10 x 96 pieces 5011120200 Waste tray IG disposable 25 pieces 5011120500 Possible suppliers for lsopropanol Carl Roth Applichem Sigma 2 Propanol 2 Propanol fur die Molekularbiologie 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no A3928 Order no 59304 1L F Order no 6752 29 InviMag Virus RNA Mini Kit IG 0515 Stratecee molecular STRATEC
30. rocessed sample volume is 200 ul elution volume 100 ul 3 Extraction of viral RNA from supernatant of stool suspension Pipet 600 ul ddH O in a 1 5 ml reaction tube not provided Add a glass stick to the stool sample and transfer the adherent sample size of a lentil in the prefilled 1 5 ml reaction tube Resuspend the sample in the prefilled water Close the tube and vortex each sample vigorously until tt becomes a homogenic suspension Centrifuge the samples for 5 min at 12 000 x g 13 400 rpm Dip carefully the pipette tip about 0 5mm below the surface and take from there 500 ul supernatant prevent the aspiration of swimming particles and transfer the sample in the sample tube and load it into the InviGenius RVIR_E100S200 processed sample volume is 200 ul elution volume 100 ul Prevention of cross contamination To comply with the demanding guidelines of in vitro diagnostics we programmed the InviGenius to route the pipettor in such a way that possible contamination risks are minimized However we recommend to apply the supplied well strios and foils beforehand and afterwards on the used wells on the unused wells of the Incubation Plate A and the Working Plate B 13 InviMag Virus RNA Mini Kit IG 0515 Preparing of the internal control for the InviGenius system Please read the instructions carefully and conduct the prepared procedure Using an internal control IC Using the InviGenius Pathogen Extraction
31. s All utilities reagents and plastics except conductive tips required for preparation of viral RNA are provided by the InviMag Virus RNA Mini Kit IG THE PRODUCT IS ITDENDED FOR USE BY PROFESSIONALS SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is validated for viral RNA extraction from cell free body fluids and rinsed liquids specifically for human serum and plasma Related applications will need a separate validation Extraction of eukaryotic RNA from samples has not been evaluated with this kit The included chemicals are only useable once Differing of starting material may lead to inoperability Therefore neither a warranty nor guarantee in this case will
32. s Ready XSDVWOtwtt User developer Figure 8 Disposable tip loading screen There are three tip rack positions on the InviGenius system Fig 10 A1 A3 corresponding to Fig 3 D1 D3 Remaining tip numbers are shown in B Tip numbers can be changed by pressing the number field directly Empty tip racks can be unloaded and reloaded by 1 Pressing the Loading Position directly The software will focus this loading position on the main screen 2 Pressing the Unload Button C 3 The loading position can be refilled with a new tip rack by pressing on the corresponding tip rack on D Each position can be filled either with 50 ul or 1100 ul filter or non filter tips However for the Virus RNA assay only 1100 ul filtered tips will be used 19 InviMag Virus RNA Mini Kit IG 0515 Attention It is very important to allocate the type of tips correctly in the software that have been loaded into the instrument In case of false tip allocation overfilling of the tip will irreparably destroy the pipettor head All protocols should be used in combination with filter tips to ensure efficient prevention of sample or reagent cross contaminations STRATEC Molecular will give no guarantee or responsibility if contaminations occur due to the use of non filtered tips Note Disposable tips are not supplied within the kit We recommend the use of validated conductive tips which can be ordered at STRATEC Molecular STRATEC Molecular offers 50 ul cond
33. sample loading rack Add the Buffers in the Buffer loading rack 200 ul sample is mixed with 400 ul Lysis Buffer RV 40 ul Proteinase K 40 ul Carrier RNA and 40 ul MAP Solution B Incubation at elevated temperature is performed for 10 min 450 ul Binding Solution is added to the lysate RNA binds to magnetic particles Magnetic separation Washing of the particle fixed viral RNA with 1 x 600 ul Wash Buffer R1 2 x 600 ul Wash Buffer R2 Magnetic separation Elution of viral RNA in 100 u Elution Buffer R Magnetic separation and removal of MAP Solution B Pure viral RNA 12 InviMag Virus RNA Mini Kit IG 0515 Preparing the samples for processing on the InviGenius system Please read the instructions carefully and conduct the prepared procedure Important Note The protocol is optimized for the isolation of viral RNA from up to 200 ul of cell free fluid To prevent possible distribution errors we highly recommend using at least 400 ul of sample in total to ensure stable processing 1 Extraction of viral RNA from serum plasma cell free body fluids This type of sample can be processed directly without any preparations Please make sure to supply at least 400 ul or dilute with PBS up to this volume RVIR_E100S200 processed sample volume is 200 ul elution volume 100 ul 2 Extraction of viral RNA from swab samples Rinse each swab with 500 ul cooled water or cooled PBS and load it into the InviGenius RVIR_E100S200 p
34. stratecee molecular ev User manual InViMag Virus RNA Mini Kit IG for use on the InviGenius STRATEC Molecular GmbH for automated purification of viral RNA from serum plasma cell free body fluids rinse liquid from swabs amp stool samples with magnetic beads 2443120100 gaa STRATEC Molecular GmbH D 13125 Berlin T Instruction for InviMag Virus RNA Mini Kit IG The InviMag Virus RNA Mini Kit IG combines the advantages of the innovative Invisorb technology with easy handling of magnetic particles for a very efficient and reliable isolation of viral RNA with high purity in a fully automated process The nucleic acid binding magnetic particles are characterized by a high specific surface area a uniform size distribution and good suspension stability Therefore the particles are highly suitable for high throughput processing of nucleic acids The InviMag Virus RNA Mini Kit IG is the ideal tool for fully walk away isolation and purification of viral RNA from 12 samples of fresh or frozen plasma serum cell free body fluids as well as rinsed liquid from swabs and supernatant from stool suspension with the InviGenius fully automated extraction system The interplay of the RNA extraction and purification chemistry provided by the InviMag Virus RNA Mini Kit IG with the InviGenius system was intensely tested C Compliance with EU Directive 98 79 EC on in vitro medical devices Not for i
35. t IG 0515 After selecting Loading the sample loading screen appears Disposable tips Loading Samples BIE SET SIE Reagents 5 Microplates Waste management Status Ready Ott ii ii User developer Assay Selection Back Figure 4 Loading screen of the InviGenius software Select Samples to proceed with the sample loading screen Samples loading SAMPLE 1 edited SAMPLE 2 edited SAMPLE 3 edited SAMPLE 4 edited SAMPLE 5 edited SAMPLE 6 edited SAMPLE edited SAMPLE 8 edited SAMPLE 9 edited SAMPLE 10 edited SAMPLE 11 edited SAMPLE 12 edited PRSPSPSKOKOKOKORORTOROROROKORORO Reagent loading Status Ready me User service Figure 5 Sample loading screen of the InviGenius software Please add the samples to the rack Please decap the tubes before transfer to the loading rack If available the primary tubes should be used directly as sample tubes If the samples are not provided in primary tubes please prepare the sample rack with primary tubes that are prefilled with samples from which the viral DNA shall be extracted Sample tubes are not provided with the kit and can be ordered at e g Sarstedt order no 55 476 5 ml tubes 75x12 mm PS or see recommendation at page 10 chapter reagents and equipment to be supplied by user For each reaction a sample volume of 200 ul is processed However it is recommended that the total sample volume filled in the sample tubes s
36. t at www sitratec com for each STRATEC Molecular product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the waste generated by the InviMag Virus RNA Mini Kit IG procedures for residual infectious materials Contamination of the waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore all waste has to be considered infectious and should be handled and discarded accordingly to local safety regulations European Community risk and safety phrases for the components of the InviMag Virus RNA Mini Kit IG to which they apply are listed below as follows Lysis Buffer RV Proteinase K warning danger H302 312 332 412 EUH032 P273 H315 319 334 335 P280 305 351 338 310 405 Wash Buffer R1 danger H302 312 332 412 EUH032 P273 H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects H315 Causes skin irritation H319 Causes serious eye irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation EUH032 Contact with acids liberates very toxic gas P273 Avoid release to the environment P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 I
37. uctive tips 10x 96 pieces order no 5011120100 and 1100 ul conductive tips 10x 96 pieces order no 5011120200 Be sure that conductive tips are used otherwise the tip detection unit installed in the pipetting unit will reject the tips and no run will be possible Disposable Sheaths Loading The sheaths are used as protection devices for the magnetic rods Disposable sheaths loading Sheath Load Position ORORORORORORORORORORORS 4 ORORKORKORKORKORKORORKORKORKORKO anes ORKORKORKORKORKORKORORKORKORKORKO OKORKOKORKOKORKORKORKORKORORO Sheath Type Microplates Status Ready X5vVtHH _UUuU lt _____0 User developer Figure 9 Disposable sheaths loading screen The loading procedure of the disposable sheaths works analogous to the disposable tip loading screen For a run always 12 disposable sheaths one row in the sheaths rack are used regardless of the processed sample numbers This is done to assure that the rods are always protected against contaminations In general the number of sheaths supplied within the kit is sufficient for the amount of runs printed on the kit package If you are lacking sheaths they can be ordered separately at STRATEC Molecular 100 pieces bulk order no 5011120300 or 10 x 48 pieces order no 5011120400 Comparable to the disposable tips loading it is possible to define the number of rows left in the tip rack by pressing on the displayed number area Mak
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