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Tissue Preparation System Extraction Protocols Online Training

Describe the extraction process and how the key reagents function during the nucleic acid extraction.  Perform a extraction protocol on the Tissue Preparation System. This clinical laboratory training qualifies for continuing education units (CEU).

The Tissue Preparation System's fully automated extraction of nucleic acids from FFPE tissue samples consists or the following: VERSANT® Tissue Preparation Reagents Tissue Preparation System Select Next to continue. Describe the nucleic acid extraction process from formalin-fixed, paraffin-embedded (FFPE) tissue sections List the key steps to run a protocol on the TPS List the three protocols of the Tissue Preparation System (TPS) State the purpose of the key reagents of the VERSANT Tissue Preparation kit Upon successful completion of this course, you will be able to: Select Next to continue. Congratulations.  You have completed the Tissue Preparation System Running a Protocal Online Training course.  Listed below are the key points that have been presented.  Take time to review the material before you proceed to the final quiz.  Describe the nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) tissue sections The VERSANT Tissue Preparation Reagents extract DNA and RNA from FFPE tissue sections. The reagents enable the automated extraction of either total nucleic acid, RNA, or both total nucleic acid and RNA in the same run. State the purpose of the key reagents of the VERSANT Tissue Preparation kit FFPE Buffer - Deparaffinizes samples and lyses the cells. Magnetic Beads - Selectively binds to substances depending on the buffer that is present in the solution. Lysis Buffer - Creates a Chaotropic, high-salt condition, that is used to capture any DNA or RNA present in the sample onto the magnetic beads. Wash Buffers 1, 2, and 3 - Removes proteins, including nucleases and other contaminants. Proteinase K - Lyses cells, digests proteins and inactivates cellular nucleases. DNase I - The inclusion or omission of the digestion step with DNase I reagents allows for subsequent analysis of either RNA or DNA, respectively. Elution Buffer - Elutes the nucleic acids in a small volume of buffer that would be ready for subsequent analysis. List the three protocols of the Tissue Preparation System The DNA Protocol (submethod) The RNA Protocol (submethod) The DNA/ RNA (split plate) Protocol (submethod) List the key steps to run a protocol on the TPS Prepare reagents and consumables Prepare tissue samples Load the deck Start a run Complete a run Select Next to continue. The VERSANT Tissue Preparation Reagents extract DNA and RNA from FFPE tissue sections. The reagents enable the automated extraction of: Total nucleic acid RNA Both total nucleic acid and RNA in the same run Extraction of nucleic acids Review a representation of the extraction process of nucleic acids from FFPE tissue.   Select the play arrow to begin the video. When complete, select the X in the upper-right corner to close the window and continue. Calibration on the CLINITEK Atlas analyzer is required any time a reagent roll with a different lot number is loaded, when a reagent roll with the same lot is loaded but the system has not been calibrated within the last 24 hours, if the system displays error messages requiring you to calibrate the system, if the system temperature changes by more than 5 degrees celsius or after hardware components are replaced and it is required by the procedure. The VERSANT Tissue Preparation Reagents kit consists of the following key reagents: FFPE Buffer Magnetic Beads Lysis Buffer Wash Buffers 1, 2, and 3 Proteinase K DNase I Elution Buffer Key Reagents of the Tissue Preparation System Learn about each of the key reagents and their function during nucleic acid extraction. Slide NumberText BlocksCalloutsAudio ScriptImage File1FFPE Buffer: Deparaffinizes samples and lyses the cells in a non-chaotropic state Storage temperature: 15–30°C Select Next to continue. Note: If audio does not automatically start, select the play arrow in the top left to begin.The FFPE buffer plays an essential role in the extraction process as it aids in the removal of paraffin wax that is used to preserve the sample. In addition, it assists in the breaking apart of the cell to release the nucleic acids into the tube.2The Magnetic Beads are an integral component in two of the critical steps of the extraction process: Negative Selection Step: Binds and eliminates any undigested tissue (debris) and proteins after lysis and prior to nucleic acid binding. Positive Selection Step: Specifically binds and isolates nucleic acids. Captures DNA or RNA present in the sample. Storage Temperature: 15–30°C Select Next to continue.    The VERSANT Tissue Preparation Reagents utilize technology based on iron oxide beads coated with a nanolayer of silica. The magnetic silica technology is used in two ways. First, it is used as a negative selection step to bind and eliminate any undigested tissue (debris) after lysis and prior to nucleic acid binding. This negative selection step allows for a standardized lysis time and ensures that all tissue debris is removed before the sample is pipetted for further processing. Second, following the lysis step, the magnetic silica technology is used to bind and isolate nucleic acids. 3Lysis Buffer: Creates a Chaotropic, high-salt condition, that is used to capture any DNA or RNA present in the sample to the magnetic beads. Storage Temperature: 15–30°C  Select Next to continue. The Lysis buffer contains a high salt concentration. This creates a Chaotropic condition which facilitates the capture of the nucleic acids present in the sample onto the magnetic beads. 4Wash buffers 1, 2, and 3 Removes proteins, including nucleases and other contaminants Wash 1: 3M Guanidine Thiocyanate (GTC), 30% Ethanol Wash 2: Buffer solution, 80% Ethanol Wash 3: Buffer solution, surfactant, and preservatives Storage Temperature: 15–30°C Select Next to continue.  Each Wash buffer has a slightly different function and chemical formulation. Wash buffer I contains GTC which aids in lysing the cells resulting in the release of the nucleic acids. Wash buffer II is used to wash away any residuals of GTC from Wash I. Wash III removes Ethanol residuals that are present in both Wash I and Wash II.5Proteinase K (PK): Lyses cells, digests proteins and inactivates cellular nucleases Storage Temperature: 2–8°C Select Next to continue.The PK works along with the FFPE and Lysis buffer to lyse the cells. It also aids in digesting the proteins and inactivating the cellular nucleases.6DNase I: The inclusion or omission of the digestion step with DNase I reagents allows for subsequent analysis of either RNA or DNA, respectively. Storage Temperature: -30° to -10°C Select Next to continue.  If DNase I is included in the protocol, it digests the DNA and purifies the RNA for RNA analysis. If DNase I is excluded from the protocol, purified total nucleic acids result for DNA analysis.7Elution Buffer: Elutes the nucleic acids in a small volume of buffer that would be ready for subsequent analysis Storage Temperature: 15–30°C When complete, select the X in the upper-right corner to close the window and continue.   The Elution Buffer is the final buffer that is added to purified nucleic acids. This buffer is compatible with real-time and end-point PCR reagents and reverse transcription enzymes. Upon successful completion of this course you will be able to: Identify the conditions under which the system will require a calibration, state the materials required for calibration, List the steps necessary to perform a calibration, locate and review the status of calibration results and identify and resolve common calibration errors. There are three extraction Protocols (sub methods) available with the Tissue Preparation Solution: DNA Protocol (sub method) RNA Protocol (sub method) DNA/RNA (split plate) Protocol (sub method) Download and print a copy of the Tissue Preparation System Extraction Protocols.   The steps required to run a protocol on the Tissue Preparation System are: Prepare reagents and consumables Prepare tissue samples Load the autoload tray Start a run Complete a run Note: Refer to the appropriate extraction protocol checklist for complete instructions when preparing a run.     Quality Control Guidelines Learn More about Quality Control guidelines for the Tissue Preparation System.   The VERSANT Tissue Preparation Reagent kit does not include quality control material. End users are responsible for validating laboratory-developed methods used in conjunction with these reagents and for implementing appropriate quality-control measures.   When complete, select the X in the upper-right corner to close the window and continue.   During routine operation of the system the LIS sends the workorders to the WAM. However, when the LIS is down, workorders can be created directly in the WAM software using the Enter Order screen.  The required fields that must be completed when entering orders are Sample ID and Test requests. The demographic information is needed only if rules are based on this information, for example, patient age or location. Select the link below to learn how to create work orders.     Key steps required to prepare reagents and consumables: Prepare reagents Vortex magnetic beads Transfer PK Prepare DNase I Load carriers/deck Preparing Reagents and Consumables Learn more about preparing reagents and consumables for a run. Checklist TitleChecklist TypeChecklist ContentPrepare Lysis and Wash 1, 2, and 3 BuffersHTML   Select each checkbox to learn more about preparing reagents and consumables. Mix the Wash 1, 2, and 3 and the Lysis Buffers by gently inverting each bottle. Pour into the corresponding troughs. Select the play arrow to begin the video.   Prepare Magnetic beadsHTML   Mix the Magnetic Beads by vortexing the bottle.  Pour into the correspondingly trough. Select the play arrow to begin the video.   Prepare FFPE and Elution BufferHTML  Mix the FFPE and the Elution Buffer by gently inverting each bottle. Pour into the corresponding troughs.   Transfer PKHTML Mix the Proteinase K by gently inverting the bottle 10 times; pour the entire volume into the labeled 5-mL tube. Note: After opening, the Proteinase K, can be stored capped at 2º to 8°C for 2 weeks. After 2 weeks, discard the tube and unused reagent. Proteinase K must be removed and stored at 2º to 8°C after each run.  Prepare DNase IHTML To prepare DNase I: Thaw the DNase I Buffer at room temperature, vortex and keep on ice until use Remove the DNase I from the freezer, mix by inversion and keep on ice until use Spin down the DNase I and DNase I Buffer Mix the thawed DNase I Buffer with the DNase I according to the table in the IFU Note: Prepare the DNase I Mix prior to each run. Prepare the DNase I Mix only for the number of samples in each run; do not store the DNase I Mix after the run. Load Carriers/DeckHTML After preparing the reagents and samples and placing them in the appropriate carriers, load the carriers onto the autoload tray of the TPS instrument. Note: Always prepare and load the carriers from left to right. When complete, select the X in the upper-right corner to close the window and continue.   Key steps required to prepare tissue samples: Prepare FFPE sample Label sample Centrifuge sample tube Transfer tube Load Sample Carrier Preparing Tissue Samples Learn about preparing tissue samples. Checklist TitleChecklist TypeChecklist ContentPrepare FFPE SampleHTML Select each checkbox to learn more about preparing tissue samples. Place each FFPE tissue section in a sterile 1.5-mL Sarstedt screw-cap micro tube. Note: The sample input for one or multiple tissue sections should not exceed a combined total thickness of 10 μm. The surface area should not exceed 600 mm2 for a single section.  Label SampleHTML   Label each sample with a sample ID. Screw down each tube cap completely. Note: The instrument does not recognize or record the sample ID information.  Centrifuge TubeHTML Centrifuge each 1.5-mL sample tube at room temperature (15º to 30°C) for 1 minute at 15,000–20,000 x g. Transfer TubeHTML Place each 1.5-mL sample tube into a 6-Tube Transport Adapter. Note: Insert sample tubes into the adapter, ensuring that the adapter snaps over the lower ring and abuts the upper ring of each sample tube.  Load Sample CarrierHTML Carefully remove the tube caps. Caution: To avoid system errors, ensure that each sample tube is placed correctly in the 6-Tube-Transport Adapter. When complete, select the X in the upper-right corner to close the window and continue.   FFPE Tissue Samples Learn more about FFPE tissue sections extracted for molecular analysis.   When complete, select the X in the upper-right corner to close the window and continue.   The Tissue Preparation System is currently validated for extraction of nucleic acids from FFPE sections with the following samples types: Surgery Specimens Needle biopsies Laser captured microdissected (LCM) cells Tissue microarray (TMA) cores Cell line blocks Siemens recommends the following storage conditions for FFPE tissue sections that will be extracted for molecular analysis: Use the original FFPE block and prepare fresh sections prior to extraction and analysis according to local procedures. Mapping Samples to Collected Eluates Compare the mapping of samples for split plate protocols and the DNA and RNA protocols. Tab TitleTextSplit Plate Mapping the samples to DNA/RNA (split) plate: The sample positions within the sample carriers map directly to the eluate collection tube positions on the Matrix 96-well plate.  DNA or RNA Mapping the samples to DNA or RNA collect tubes: The sample positions with the sample carriers map directly to the eluate tube collection positions in the eluates carriers. When complete, select the X in the right-hand corner to close the window and continue.    Loading the autoload tray is an essential part of running the Tissue Preparation System. The reagents and consumables are first loaded onto the carriers, which are then loaded onto the autoload tray. Download and print a copy of the Tissue Preparation System Deck Layout.   Loading the Autoload Tray Learn how to load the autoload tray.   Select the play arrow to begin the video. When complete, select the X in the upper-right corner to close the window and continue. Navigate the urinalysis WAM software by selecting an item listed in a drop down menu on the title bar or by selecting a fast button icon. The menu panel provides tools to exit screens, save selections or exit the WAM software. Function keys are also used to perform specific operations. Select next to continue. Starting a run on the Tissue Preparation System requires operating the Hamilton MICROLAB® STARlet software. Select the MICROLAB star run icon to execute a protocol. Starting a Run Learn how to start a run in the Hamilton MICROLAB® STARlet software. Instructions:If media does not automatically start, select the play arrow to begin.Flash File:/content/generator/Course_90004512/sim_TPS_RunningAProtocol2_800x600/sim_TPS_RunningAProtocol2_800x600.htmHTML5 File:/content/generator/Course_90004512/sim_TPS_RunningAProtocol2_HTML5_800x600/index.htmlPDF File: The Eluates from the DNA/RNA (split) Protocol (sub method) are pipetted by the System into a Matrix plate. The Eluates from a DNA or RNA Protocol (sub method) are pipetted by the System into Eluate collection tubes.   These Eluates can be used for various downstream applications and are compatible with real-time and end-point PCR, reverse transcription (RT), polymorphism analysis, microarray analysis, and sequencing.   Completing a Run Compare the completion of a DNA/RNA split method to that of a DNA or RNA method. Tab TitleTextDNA/RNA Split Submethod DNA/RNA split submethod: Seal the 0.75-mL Matrix 96-well plate with the Matrix SepraSeal Cap Mats when the extraction is complete Position the caps over the tubes, then press firmly and evenly over the entire plate Analyze the results directly or store the extracted samples (purified eluates) at     -80ºC until used DNA or RNA Submethod DNA or RNA submethod: Firmly cap the eluate collection tubes when extraction is complete.  Analyze the results directly or store the extracted samples (purified eluates) at -80ºC until used. When complete, select the X in the upper-right corner to close the window and continue.

  • tissue
  • gene
  • extract
  • protocols