Isolation of Total RNA from Whole Blood and from Cells Isolated from Whole Blood

Transcription

1 Isolation of Total RNA from Whole Blood and from Cells Isolated from Whole Blood Protocol DRAFT August 27, :39 pm, BloodRNA_TitlePg.fm

2 Copyright 2004, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: PLEASE REFER TO THE ABI PRISM 6100 NUCLEIC ACID PREPSTATION AND ABI PRISM 6700 AUTOMATED NUCLEIC ACID WORKSTATION USER S MANUALS AND THE ABSOLUTERNA WASH SOLUTION PACKAGE INSERT FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. ABI PRISM and its Design and Applied Biosystems are registered trademarks and AB (Design), ABI, and Applera are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. Part Number Rev. B 08/2004 DRAFT August 27, :39 pm, BloodRNA_TitlePg.fm

3 Contents Section 1: Introduction Overview Advantages of Using Applied Biosystems Total RNA Chemistry Yields of Total RNA Derived from Blood Stability of Isolated Total RNA Section 2: Materials, Safety, and Technical Support Materials Required but Not Supplied Safety Technical Support Section 3: Lysis Biohazard Lysing Fresh Whole Blood or Isolated Blood Cell Samples Storing the Lysate Section 4: About the Ultra-Low gdna Protocol Using AbsoluteRNA Wash Solution Overview Amplification Examples Section 5: Purification Using the 6700 Workstation Introduction Creating a New RNA Archive Protocol Performing the Purification Run Section 6: Purification Using the 6100 PrepStation Introduction Accessing a Method Performing the Purification Run i

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5 Section 1: Introduction Overview About This Protocol This protocol explains how to use Applied Biosystems total RNA chemistry and nucleic acid purification platforms to isolate total RNA from: Whole blood Cells isolated from whole blood, for example: Lymphocytes Leukocytes Peripheral blood mononuclear cells (PBMCs, also called buffy coats ) IMPORTANT Isolated blood cell samples can be processed using this protocol with the proviso that these samples are isolated from fresh blood and not frozen before cell lysis occurs. About Total RNA Chemistry Applied Biosystems total RNA chemistry uses a unique formulation to effectively lyse whole blood or isolated blood cells. Lysis occurs almost immediately after the reagent is added to the blood sample. The reagent inactivates cellular RNases, preventing degradation of the isolated nucleic acid while RNA is selectively precipitated; gdna and proteins remain in the solution. This allows a separation of total RNA from gdna and other cellular debris. The blood lysate is then transferred to the total RNA purification tray, where it is passed through the tray under precisely controlled vacuum conditions. The total RNA purification tray consists of: 96 wells (650 µl/well) A membrane to physically capture the precipitated RNA A low-retention-volume drip director An aerosol guard to prevent well-to-well cross-contamination 1

6 96-Well Format Instrument Systems Summary Diagram Applied Biosystems total RNA chemistry and instrument systems enable purification of 96 samples of total RNA from whole blood in 1 hour or less. The purification procedures discussed in this document can be performed on the following Applied Biosystems instrument systems: ABI PRISM 6700 Automated Nucleic Acid Workstation (6700 Workstation) ABI PRISM 6100 Nucleic Acid PrepStation (6100 PrepStation) The procedures required to isolate RNA from whole blood or isolated blood cells are summarized in the diagram below. Remove blood from the organism. Lyse the blood samples immediately (within 1 h). Optional. Store the blood lysate: Store at 4 C for short-term storage OR Freeze at -20 to -80 C for long-term storage Choose the appropriate purification protocol: Standard RNA isolation protocol OR Ultra-low gdna protocol using AbsoluteRNA Wash Solution Perform purification using the: 6100 PrepStation OR 6700 Workstation 2

7 Advantages of Using Applied Biosystems Total RNA Chemistry Overview High-quality RNA purified from whole blood or isolated blood cells is required for many of today s gene expression studies using real-time polymerase chain reaction (PCR) or micro-arrays. The RNA obtained for these studies must be highly pure, since accuracy and sensitivity are critical for interpretation of results. Applied Biosystems has developed new techniques for producing the highest quality total RNA from whole blood or isolated blood cells. By using Applied Biosystems total RNA chemistry and instrument systems, you can: Use blood samples from a variety of species (including human). Isolate highly pure and highly stable total RNA from whole blood directly, using any common anticoagulant. Eliminate sample pretreatments (e.g., proteinase K digestion or red blood cell lysis). Use a 96-well format for high throughput. Use all other Applied Biosystems existing total RNA reagents and consumables. Blood Samples from a Variety of Species Blood samples can be taken from a variety of species, including human. Yields of RNA vary considerably from animal to animal and will also depend on the health of the animal. (See Yields of Total RNA Derived from Blood on page 5.) Note Protocols and techniques for taking blood samples from animals or patients are beyond the scope of this document and will not be discussed here. Purity Automated RNA purification from whole blood or isolated blood cells is a formidable challenge. Blood contains high levels of: RNases Multiple PCR inhibitors (e.g., heme, immunoglobulin G, and lactoferrin) Protein Genomic DNA (gdna) 3

8 RNases and PCR inhibitors plague existing extraction procedures and downstream assays. Contaminating gdna may affect the accuracy of RT-PCR and other downstream procedures. Any automated procedure used to purify RNA from blood must address these factors and provide material that is highly pure. Applied Biosystems blood RNA isolation protocol generates total RNA that is free of RNases, free of PCR inhibitors, and has low to zero levels of contaminating gdna. About Anticoagulants Blood samples collected with the anticoagulant, heparin, may also inhibit PCR. All three common types of blood anticoagulants (citrate, EDTA, and heparin) are compatible with Applied Biosystems blood RNA isolation protocol. Removing Residual DNA from Isolated Total RNA An optional wash solution to remove DNA and heparin inhibition can be applied as an additional step in this protocol. For more information, see About the Ultra-Low gdna Protocol Using AbsoluteRNA Wash Solution on page 23. Stability of Isolated RNA Eliminating Sample Pretreatments RNA isolated using Applied Biosystems total RNA chemistry is highly stable. (See Yields of Total RNA Derived from Blood on page 5.) A 260/280 ratios of isolated RNA are routinely demonstrated to be >1.9 and are typically close to the theoretically pure limit of absorbance ratio of A 260/280 = 2.1. This means that the RNA is free from the majority of contaminating proteins, including RNases. Many of the currently available protocols for isolating total RNA from blood incorporate a leukocyte isolation process by either centrifugation or a selective red blood cell (RBC) lysis protocol. However, these processes either add cost or are time and labor intensive. In addition, these processes do not neutralize biohazardous agents in the samples and require manual manipulation. Preventing RNase activity from degrading RNA in blood lysates also is a difficult task, as the RNase activity in blood is much higher than in other tissues. A much simpler, safer, and less time-consuming method involves the direct lysis of whole blood. Applied Biosystems total RNA isolation 4

9 protocol lyses blood directly and allows for fast, high-throughput purification. Yields of Total RNA Derived from Blood Blood Samples from a Variety of Species As shown in Figure 1, Applied Biosystems total RNA chemistry can be used across a wide variety of species to generate high-quality, intact, total RNA. The RNA concentration obtained varies depending on the animal species. In this experiment, 1 volume of phosphate buffered saline (PBS) and 2 volumes of 2X Nucleic Acid Purification Lysis Solution (P/N ) were added to 1 volume of whole fresh (never frozen) blood from either mouse, rat, rabbit, human, pig, cow, or horse, and thoroughly mixed. A minimum of 1 ml/well of these blood lysates was processed for total RNA on the 6100 PrepStation using standard RNA isolation protocols for this platform. Approximately 2 µg of RNA were used for each lane of the gel. Samples were analyzed in duplicate. Figure 1 Total RNA isolation from whole fresh blood from a variety of species; the lane designated M is a molecular weight marker 5

10 Yields Across a Variety of Species Figure 2 shows the yields of total RNA obtained from a variety of species. The yields are expressed as µg of RNA isolated per ml of whole blood. The approximate weight of the animal in kilograms is also shown. RNA from Blood µ g/ml Blood mouse rat 0.2 rabbit 4 pig 250 cow 450 horse 750 human 70 Animal (kg) Figure 2 Yield of total RNA from a variety of species 6

11 Stability of Isolated Total RNA Overview The presence of contaminating proteins in most RNA preparations is probably responsible for the perceived instability of RNA at room temperature. RNA isolated with large quantities of protein almost certainly contains residual RNase activity, which contributes to degradation when the samples are thawed and held at room temperature for significant lengths of time. Care should therefore be taken to ensure that total RNA is as free from protein as possible. Applied Biosystems total RNA chemistry removes virtually all proteins and provides highly pure, highly stable total RNA. Using the Absorbance Ratio to Determine Protein Contamination Nucleic acids have an absorption maxima in the ultraviolet wavelength of 260 nm, while proteins have a maxima of 280 nm. The ratio of these numbers can therefore provide a guide to the level of protein contamination in isolated total RNA. Theoretically pure total RNA has an A 260/280 of 2.1. However, care should be taken when interpreting A 260/280 ratios. A 260/280 ratios of 1.8 to 1.9 can indicate up to 80% by weight of protein contamination in the RNA sample. It is thought that this protein background is the major cause of PCR and reverse transcription inhibition as well as thermal instability at room temperature in sub-optimally purified RNA. In the experiment shown in Figure 3 on page 8, RNA was first purified and tested to ensure that the material was protein free. It was then spiked with the common protein Bovine Serum Albumin (BSA) and the A 260/280 ratio was measured at different percentages of protein contamination. The graph shows that RNA samples of A 260/280 ratios of 1.8 and below may contain up to 80% by weight of protein. 7

12 2.1 j 2 A260/280 Ratio Figure % Protein (BSA) A 260/280 ratio versus percentage of protein in Raji RNA Total RNA Chemistry Removes Contaminating Proteins When obtained using Applied Biosystems total RNA chemistry, RNA is highly pure, free from contaminating proteins and metal ions. This allows the RNA to remain stable for a number of hours at room temperature and after incubation at 37 C. In the experiment shown in Figure 4 on page 9, four samples of a 2-mL blood/pbs/1x Lysis Buffer solution (0.5 ml of whole human blood total) were processed using Applied Biosystems total RNA chemistry. Two of the isolated and purified RNA samples were allowed to stand overnight at room temperature; the other two samples were incubated at 37 C for 3 hours. Approximately 2 µg of RNA were used for each lane of the gel. The samples were analyzed in duplicate. The gels show that when RNA is isolated in a process that removes the majority of protein contamination, RNA is a relatively stable molecule. 8

13 Incubated overnight at room temp. Incubated for 3 h at 37 C 28S rrna 18S rrna Figure 4 Denaturing agarose gel images of total RNA from human blood 9

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15 Section 2: Materials, Safety, and Technical Support Materials Required but Not Supplied Overview The following tables list the equipment, accessories, and chemicals required to perform this protocol. Unless otherwise noted, many items listed can be obtained from a major laboratory supplier (MLS). Equipment Equipment ABI PRISM 6700 Automated Nucleic Acid Workstation OR ABI PRISM 6100 Nucleic Acid PrepStation Microcentrifuge Pipettors Vortexer Source See your Applied Biosystems sales representative MLS MLS MLS Accessories Accessories Supplier Part Number Deep-well plate Applied Biosystems 96-Well Optical Reaction Plate with Barcode (also called archive plate and reaction plate ) Splash guard Total RNA purification tray Applied Biosystems Applied Biosystems Applied Biosystems Additional Accessories for Purification on the 6700 Workstation Archive covers Applied Biosystems Conductive pipette tips, 1000-µL Applied Biosystems

16 Accessories Supplier Part Number Conductive pipette tips, 200-µL Reagent reservoirs, 120-mL Note This product comes with a sheet of barcode labels for Applied Biosystems nucleic acid purification reagents. Applied Biosystems Applied Biosystems Chemicals Chemical Supplier Part Number Phosphate buffered saline (PBS), MLS calcium/magnesium-free 2X Nucleic Acid Purification Lysis Solution Applied Biosystems IMPORTANT This is supplied as a 2X formulation and may be used at the 2X formulation for sample storage. Otherwise, it should always be used at a final concentration of 1X (referred to as 1X Lysis Buffer ). RNA Purification Wash Solution 1 RNA Purification Wash Solution 2 Nucleic Acid Purification Elution Solution AbsoluteRNA Wash Solution Applied Biosystems Applied Biosystems Applied Biosystems Applied Biosystems

17 Safety Documentation User Attention Words Five user attention words appear in the text of all Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below. Note Calls attention to useful information. IMPORTANT Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical.! CAUTION Indicates a potentially hazardous situation which, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices.! WARNING Indicates a potentially hazardous situation which, if not avoided, could result in death or serious injury.! DANGER Indicates an imminently hazardous situation which, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. Chemical Hazard Warning! WARNING CHEMICAL HAZARD. Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury, illness, or death. Read and understand the material safety data sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (e.g., fume hood). For additional safety guidelines, consult the MSDS. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer s cleanup procedures as recommended on the MSDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. 13

18 Chemical Waste Hazard Warning! WARNING CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury, illness, or death. Read and understand the material safety data sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. Handle chemical wastes in a fume hood. Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (e.g., fume hood). For additional safety guidelines, consult the MSDS. After emptying the waste container, seal it with the cap provided. Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations. Site Preparation and Safety Guide About MSDSs A site preparation and safety guide is a separate document sent to all customers who have purchased an Applied Biosystems instrument. Refer to the guide written for your instrument for information on site preparation, instrument safety, chemical safety, and waste profiles. Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer. When hazards exist, warnings are prominently displayed on the labels of all chemicals. Chemical manufacturers supply a current material safety data sheet (MSDS) before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update. MSDSs provide you with the safety information you need to store, handle, transport and dispose of the chemicals safely. We strongly recommend that you replace the appropriate MSDS in your files each time you receive a new MSDS packaged with a hazardous chemical.! WARNING CHEMICAL HAZARD. Be sure to familiarize yourself with the MSDSs before using reagents or solvents. 14

19 Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below. To order documents by automated telephone service: 1 From the U.S. or Canada, dial Follow the voice instructions to order documents (for delivery by fax). Note There is a limit of five documents per fax request. To order documents by telephone: In the U.S. Dial , and press 1. In Canada Dial , and press 1 for English or 2 for French. To view, download, or order documents through the Applied Biosystems web site: 1 Go to 2 Click SERVICES & SUPPORT at the top of the page, click Documents on Demand, then click MSDS. 3 Click MSDS Index, search through the list for the chemical of interest to you, then click on the MSDS document number for that chemical to open a PDF version of the MSDS. For chemicals not manufactured or distributed by Applied Biosystems, call the chemical manufacturer. 15

20 Technical Support Obtaining Services and Support For services and support, access the Applied Biosystems Web site: At the Applied Biosystems Web site, you can: Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition, the Applied Biosystems Web site provides a list of telephone and fax numbers that can be used to contact Technical Support. 16

21 Section 3: Lysis Biohazard Warning The procedures in this section discuss handling of blood samples. Before performing these procedures, please read and follow the biohazard warning below.! WARNING BIOHAZARD. Biological samples such as tissues and blood have the potential to transmit infectious diseases. Follow the U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no ) and in Occupational Safety and Health Standards, Toxic and Hazardous Substances (29 CFR ) concerning the principles of risk assessment, biological containment, and safe laboratory practices for activities involving clinical specimens. You can obtain additional information by connecting to the government Web site Lysing Fresh Whole Blood or Isolated Blood Cell Samples When to Lyse Samples Fresh whole blood or isolated blood cell samples should be lysed as quickly as possible after being drawn from the organism (within 1 hour). If you will not be lysing the samples, freeze them within 1 hour of drawing. IMPORTANT Freezing the whole blood or isolated blood cell sample, thawing, and then lysing, WILL result in decreased RNA yields in this protocol. 17

22 Effects of Prolonged Storage at Room Temperature Prolonged storage of fresh, unlysed whole blood or isolated blood cells at room temperature may affect the expression levels of a number of important gene targets. In the experiment shown in Figure 5 and Figure 6 on page 19, whole human blood was drawn and stored under one of the following conditions: Temperature Time 4 C 1 hour 4 hours Overnight 18 to 23 C (ambient) 1 hour 4 hours Overnight The blood sample was then lysed using Applied Biosystems total RNA chemistry and purified using the standard RNA protocol on the 6100 PrepStation. Relative gene expression across the different storage conditions was then analyzed by a one-step RT-PCR protocol using 85 different gene targets (derived from Applied Biosystems Pre-Developed Assay Reagents list). The list of 85 targets included 11 endogenous control or housekeeping genes. For some gene targets (e.g., interleukin-8 [IL-8], c-myc, CCR-3, and MCP-2), there was significant up and down regulation by as much as a factor of 10 for samples stored at ambient conditions for more than 1 hour. 18

23 Relative Expression IL-7 LT-beta FAS FasL p53 CCR-3 MCP degrees 1 hr 4 degrees 4 hr 4 degrees O/N Ambient 1 hr Ambient 4 hr Ambient O/N Figure 5 Genes down-regulated Relative Expression IL-1 alpha IL-8 c-myc c-fos G-CSF MCP-1 MIP-1-alpha degrees 1 hr 4 degrees 4 hr 4 degrees O/N Ambient 1 hr Ambient 4 hr Ambient O/N Figure 6 Genes up-regulated 19

24 Lysing Whole Blood To lyse whole blood: Step Action 1 Add 1 volume of calcium/magnesium-free PBS to 1 volume of fresh (never frozen) whole blood. For example, add 5 ml of PBS to 5 ml of fresh whole blood. 2 Add an equal volume of 2X Nucleic Acid Purification Lysis Solution (P/N ) to the diluted blood sample. For example, add 10 ml of 2X Nucleic Acid Purification Lysis Solution to the 10 ml of diluted blood sample obtained in step 1.! WARNING CHEMICAL HAZARD. Nucleic Acid Purification Lysis Solution causes eye, skin, and respiratory tract irritation. It is harmful if swallowed. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Lysing Isolated Blood Cell Pellets Making 1X Lysis Buffer IMPORTANT The 2X Nucleic Acid Purification Lysis Solution (P/N ) must be used at a final concentration of 1X when lysing isolated blood cell pellets. This is referred to as 1X Lysis Buffer. To make 1X Lysis Buffer: Step Action 1 Dilute 1 volume of 2X Nucleic Acid Purification Lysis Solution (P/N ) with 1 volume of calcium/magnesium-free PBS.! WARNING CHEMICAL HAZARD. Nucleic Acid Purification Lysis Solution causes eye, skin, and respiratory tract irritation. It is harmful if swallowed. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 2 Mix by gently inverting the tube 3 or 4 times. 20

25 Lysing Isolated Blood Cell Pellets To lyse isolated blood cell pellets: Step Action 1 Isolate cells from fresh blood per your laboratory s normal procedures or per the manufacturer s instructions. IMPORTANT Isolated blood cell samples can be processed using this protocol with the proviso that these samples are isolated from fresh blood and not frozen before cell lysis occurs. 2 Lyse the isolated blood cell pellets at a maximum concentration of: 1 x 10 6 cells per 200 µl of 1X Lysis Buffer (page 20) IMPORTANT The maximum input level of isolated blood cells for the total RNA purification tray is 1 x 10 6 cells per well. Input levels above this may cause the purification tray to clog. If this happens, you will not be able to complete the protocol. In addition, input levels above 1 x 10 6 cells per 200 µl of 1X Lysis Buffer may result in isolation of degraded RNA due to endogenous RNase activity. Storing the Lysate When Storage Is Required Blood lysate should not be allowed to reach room temperature as this may significantly impact RNA recovery. If you will not be purifying the blood lysate for RNA immediately, store the blood lysate per the procedure below. Storing the Lysate To store the lysate: Step Action 1 Lyse fresh whole blood or isolated blood cell samples immediately (within 1 h) upon drawing, per the procedures on pages 17 to

26 To store the lysate: (continued) Step Action 2 If you will be storing the lysate... Then store at h 4 C >12 h -20 to -80 C. Note We recommend -80 C for extended storage periods or archival purposes (i.e., >3 months). 22

27 Section 4: About the Ultra-Low gdna Protocol Using AbsoluteRNAWash Solution Overview When to Use the Ultra-Low gdna Protocol The ultra-low gdna protocol is identical to the standard RNA isolation protocol, except for the simple addition of AbsoluteRNA Wash Solution. By using AbsoluteRNA Wash Solution, residual gdna can be removed from total RNA. If very low to zero levels of contaminating gdna are required for your application, use the ultra-low gdna protocol rather than the standard RNA isolation protocol. Note Procedures for both the ultra-low gdna protocol and the RNA isolation protocol begin on page 28 (for the 6700 Workstation) or page 38 (for the 6100 PrepStation). About AbsoluteRNA Wash Solution AbsoluteRNA Wash Solution is an optimized, one-tube combination of DNase enzyme and buffer. The solution is supplied ready-to-use in a 10-mL vial, which is sufficient for 100 to 200 wells of isolated total RNA. IMPORTANT AbsoluteRNA Wash Solution should be thawed before use. Do not re-freeze more than three times. If necessary, aliquot the solution into smaller vials and store as recommended. About the Automated Process The automated process for removing gdna with AbsoluteRNA Wash Solution is as follows: A 50- or 100-µL aliquot of AbsoluteRNA Wash Solution is added to each purification tray well. A 15-minute incubation is performed, which digests the remaining low levels of gdna. A volume of RNA Purification Wash Solution 2 is added. A further 5-minute incubation is performed. The RNA isolation protocol then continues as normal. 23

28 As shown in Figure 7 on page 24, addition of AbsoluteRNA Wash Solution is performed after the RNA is captured on the membrane of the total RNA purification tray and between additions of RNA Purification Wash Solution 2. Add 2X Nucleic Acid Purification Lysis Solution and process sample Whole blood/isolated blood cell lysate Prewet purification tray Add lysate to purification tray Vacuum filtration Add RNA Purification Wash Solution 1 Vacuum filtration Add RNA Purification Wash Solution 2 Vacuum filtration Add AbsoluteRNA Wash Solution 15-min incubation Add RNA Purification Wash Solution 2 Add RNA Purification Wash Solution 2 5-min incubation Vacuum filtration Vacuum filtration Add Nucleic Acid Purification Elution Solution Vacuum filtration Figure 7 Flowchart of the ultra-low gdna protocol 24

29 Amplification Examples Amplification of Human Total RNA In this experiment, human total RNA was isolated from 96, 0.5-mL human whole blood samples using the 6100 PrepStation. A gdna removal step was incorporated using AbsoluteRNA Wash Solution. The total RNA was diluted 1:50 and amplified in a 5 nuclease- based, one-step RT-PCR TaqMan assay for the cyclophilin amplicon on the ABI PRISM 7700 Sequence Detection System. Figure 8 shows that for the 78 samples assayed, the yield of RNA was highly reproducible from well to well. Wells 1 to 15: Human control RNA standard curve, 5 ng/µl to ng/µl Wells 16 to 19: No template controls Wells 20 to 96: Samples Figure 8 Human cyclophilin quantitative RT-PCR: amplification plot of C T versus well position for 0.5 ml of whole blood (1:50) dilution 25

30 Amplification of Contaminating gdna Figure 9 shows the same assay as that outlined in Figure 8 on page 25, but the amplification reaction is performed without reverse transcriptase (i.e., only contaminating gdna carried through the total RNA isolation step is being amplified in this reaction). Wells 1 to 12: Human control gdna standard curve Wells 13 to 16: No template controls Wells 17 to 65: Samples treated with AbsoluteRNA Wash Solution and amplified for contaminating gdna Wells 65 to 96: Samples not treated with AbsoluteRNA Wash Solution and amplified for contaminating gdna Figure 9 Human cyclophilin RT-minus assay: amplification plot of C T versus well position for 0.5 ml of whole blood (1:50) dilution 26

31 Section 5: Purification Using the ABI PRISM 6700 Automated Nucleic Acid Workstation Introduction Overview The 6700 Workstation is an automated nucleic acid purification platform. Once you place the lysate into the 6700 Workstation, the 6700 Workstation automatically performs all other operations. The purification run on the 6700 Workstation consists of the following procedures: Procedure See Page Creating a New RNA Archive Protocol 28 Performing the Purification Run 31 Biohazard Warning The procedures in this section discuss handling of blood samples. Before performing these procedures, please read and follow the biohazard warning below.! WARNING BIOHAZARD. Biological samples such as tissues and blood have the potential to transmit infectious diseases. Follow the U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no ) and in Occupational Safety and Health Standards, Toxic and Hazardous Substances (29 CFR ) concerning the principles of risk assessment, biological containment, and safe laboratory practices for activities involving clinical specimens. You can obtain additional information by connecting to the government Web site Laser Hazard Warning The procedures in this section require the use of a barcode reader to load the consumables and reagents onto the deckspace. Please read and follow the laser hazard warning below. 27

32 ! WARNING LASER HAZARD. Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots. Never look into the laser beam. Remove jewelry and anything else that can reflect the beam into your eyes. Protect others from exposure to the beam. Creating a New RNA Archive Protocol Two Protocols Available There are two protocols available for performing total RNA purification from blood on the 6700 Workstation: Standard RNA isolation protocol The standard RNA isolation protocol should give total RNA with <0.5% by weight of contaminating gdna. Ultra-low gdna protocol This protocol includes a gdna removal step using AbsoluteRNA Wash Solution (P/N ). If very low to zero levels of contaminating gdna are required for your application, use the ultra-low gdna protocol. To create either protocol, see below. Creating a New RNA Archive Protocol To create either the standard or ultra-low gdna protocol: Step Action 1 Log in to the 6700 Workstation and select the Protocol tab. 2 In the Protocol section, click the New button under the RNA/DNA Archive checkbox. Click this button The New RNA/DNA Archive Protocol dialog box opens. 28

33 To create either the standard or ultra-low gdna protocol: (continued) Step 3 Action If you are creating the... standard RNA isolation protocol Then... enter the parameters shown below. Cont d 29

34 To create either the standard or ultra-low gdna protocol: (continued) Step 3 Cont d Action If you are creating the... ultra-low gdna protocol Then... enter the parameters shown below. Note This protocol includes a gdna removal step using AbsoluteRNA Wash Solution (P/N ). This step adds an additional 20 min to the purification process. 4 Click OK to save the protocol, then continue with Performing the Purification Run on page

35 Performing the Purification Run Overview For blood lysate, you perform a purification run on the 6700 Workstation as you normally would. That is, you: Load the samples Make selections on the Protocol tab Set up the deckspace Start the run The parameters and reagents specific to blood lysate are provided in the procedure below. For More Information The procedure below provides a broad overview of the steps required to perform a purification run on the 6700 Workstation. If you need more detailed procedures, refer to the ABI PRISM 6700 Automated Nucleic Acid Workstation User s Manual (P/N ). Performing a Purification Run for Blood Lysate To perform a purification run for blood lysate: Step Action Load the Samples 1 Pipette the blood lysate into a deep-well plate. Note The maximum volume of blood lysate that the total RNA purification tray can accommodate is 600 µl. 31

36 To perform a purification run for blood lysate: (continued) Step Action 2 Place the deep-well plate at the secondary input position, as shown below. The deep-well plate is designed to sit on three of the four pins at the secondary input position. The top right corner of the plate is notched to fit AGAINST the top right pin, not OVER it. Secondary input position Location pins Deep-well plate Note If the pipette tips do not enter the wells of the deep-well plate correctly, remove the two captive screws from the plate and rotate the plate 180 degrees. Firmly re-attach the captive screws before continuing. Make Selections on the Protocol Tab 3 Log in to the 6700 Workstation and select the Protocol tab. 4 Check the box next to the new RNA Archive protocol, which was created as described on page Select Lysed in the Input Plate Type pop-up menu. 6 Populate the sample list to show how many samples are to be run. 7 In the Deckspace tab, verify the protocol setup. If the Deckspace tab becomes active, the protocols are set up properly. Otherwise, resolve any errors before proceeding. Refer to the ABI PRISM 6700 Automated Nucleic Acid Workstation User s Manual if necessary. Set Up the Deckspace 8 In the Instrument tab, click the Cool Peltiers button. 32

37 To perform a purification run for blood lysate: (continued) Step Action 9 Assemble the consumables and reagents required, as listed below. Consumables and Reagents Splash guard 96-Well Optical Reaction Plate with Barcode Conductive pipette tips, 1000-µL Conductive pipette tips, 200-µL Nucleic Acid Purification Elution Solution Reagent reservoirs, 120-mL RNA Purification Wash Solution 1 RNA Purification Wash Solution 2 Total RNA purification tray Deep-well plate AbsoluteRNA Wash Solution Note AbsoluteRNA Wash Solution is only required for the ultra-low gdna protocol. 10 In the Deckspace tab, load the plates on the deckspace. Deckspace Location Input 1 Archive Purification Filtrate Dilution 1 Dilution 2 Output 1 Output 2 Output 3 Output 4 Plate Required Placeholder 96-well plate 96-Well Optical Reaction Plate with Barcode Total RNA purification tray Deep-well plate Placeholder 96-well plate Placeholder 96-well plate Placeholder 96-well plate Placeholder 96-well plate Placeholder 96-well plate Placeholder 96-well plate 33

38 To perform a purification run for blood lysate: (continued) Step Action 11 Load the tips on the deckspace. Deckspace Location Tips 1 to 4 Tip 5 to 8 Tip Type 200-µL disposable 1000-µL or 200-µL disposable 12 Fill the reagent reservoirs and load them on the deckspace. For the standard RNA isolation protocol, use: Reagent Barcode label Position RNA Purification Wash Wash Solution 1 1 Solution 1 RNA Purification Wash Wash Solution 2 2 Solution 2 RNA Purification Wash Wash Solution 2 3 Solution 2 Nucleic Acid Purification Elution Solution 8 Elution Solution For the ultra-low gdna protocol, use the reagents above, plus: AbsoluteRNA Wash None 4 Solution RNA Purification Wash Solution 2 Wash Solution 2 2! CAUTION CHEMICAL HAZARD. RNA Purification Wash Solution 1 may cause eye, skin, and respiratory tract irritation. Contact with acids or bleach liberates toxic gases. DO NOT ADD acids or bleach to any liquid waste containing RNA Purification Wash Solution 1. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.! WARNING CHEMICAL HAZARD. RNA Purification Wash Solution 2 is a flammable liquid and vapor. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 13 Load the splash guard on the deckspace. 34

39 To perform a purification run for blood lysate: (continued) Step Action 14 Verify the deckspace, close the instrument door, and start the purification run. 35

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41 Section 6: Purification Using the ABI PRISM 6100 Nucleic Acid PrepStation Introduction Overview The 6100 PrepStation is a semi-automated nucleic acid purification platform. The firmware and an integrated vacuum system automatically perform all vacuum operations. You must add all samples and wash reagents. The purification run on the 6100 PrepStation consists of the following procedures: Procedure See Page Accessing a Method 38 Performing the Purification Run 40 Biohazard Warning The procedures in this section discuss handling of blood samples. Before performing these procedures, please read and follow the biohazard warning below.! WARNING BIOHAZARD. Biological samples such as tissues and blood have the potential to transmit infectious diseases. Follow the U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no ) and in Occupational Safety and Health Standards, Toxic and Hazardous Substances (29 CFR ) concerning the principles of risk assessment, biological containment, and safe laboratory practices for activities involving clinical specimens. You can obtain additional information by connecting to the government Web site 37

42 Accessing a Method Two Protocols (Methods) Available There are two protocols (methods) available for performing total RNA purification from blood on the 6100 PrepStation: Standard RNA isolation protocol (RNA Blood method) The standard RNA isolation protocol should give total RNA with <0.5% by weight of contaminating gdna. Ultra-low gdna protocol (RNA Blood-DNA method) This protocol includes a gdna removal step using AbsoluteRNA Wash Solution (P/N ). If very low to zero levels of contaminating gdna are required for your application, use the ultra-low gdna protocol. To access either method, see below. Accessing the RNA Blood or RNA Blood-DNA Method To access either the RNA Blood or RNA Blood-DNA method: Step Action 1 In the main menu, press F3 (User). The Select User Name screen opens. Select User Name <ABI> markh <ALL> markr andy peterh markb Select New Edit Delete Cancel F1 F2 F3 F4 F5 2 Use the arrow keys to highlight user ABI. 3 Press F1 (Select). The main menu displays ABI as the user name. HH:MM:SS Applied Biosystems MM:DD:YY ABI PRISM 6100 PrepStation Version Quick User: <ABI> Method User Log Util F1 F2 F3 F4 F5 38

43 To access either the RNA Blood or RNA Blood-DNA method: (continued) Step Action 4 Press F2 (Method). The Method Select 1 screen opens. Method User Steps LastUsed Pre-Filter ABI 3 01/16/01 RNA Blood ABI 9 01/15/01 RNA Cell ABI 9 01/04/01 RNA Tissue-Filtr ABI 7 01/17/01 RNA Blood-DNA ABI 9 01/17/01 Run New F1 F2 F3 F4 F5 5 If you are performing the... standard RNA isolation protocol ultra-low gdna protocol Edit Then... More Done a. Use the up and down arrow keys to highlight the RNA Blood method. b. Continue with Performing the Purification Run on page 40. a. Use the up and down arrow keys to highlight the RNA Blood-DNA method. b. Continue with Performing the Purification Run on page

44 Performing the Purification Run Overview For blood lysate, you perform a purification run on the 6100 PrepStation as you normally would. That is, you: Load the disposables Load the total RNA purification tray Run the method The parameters and reagents specific to blood lysate are provided in the procedure below. For More Information The procedure below provides a broad overview of the steps required to perform a purification run on the 6100 PrepStation. If you need more detailed procedures, refer to the ABI PRISM 6100 Nucleic Acid PrepStation User s Manual (P/N ). Performing a Purification Run for Blood Lysate To perform a purification run for blood lysate: Step Action Load the Disposables 1 Place the following disposables on the 6100 PrepStation as indicated: a. 96-Well Optical Reaction Plate with Barcode (P/N ) in the collection compartment b. Splash guard (P/N ) in the waste compartment c. Total RNA purification tray (P/N ) in the carriage 2 Move the carriage to the waste position. Push the carriage handle down until it locks into position (seals). Load the Total RNA Purification Tray 3 Prewet the purification tray by pipetting 40 µl of RNA Purification Wash Solution 1 over each well of the purification tray.! CAUTION CHEMICAL HAZARD. RNA Purification Wash Solution 1 may cause eye, skin, and respiratory tract irritation. Contact with acids or bleach liberates toxic gases. DO NOT ADD acids or bleach to any liquid waste containing RNA Purification Wash Solution 1. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 40

45 To perform a purification run for blood lysate: (continued) Step Action 4 Add the lysate to the total RNA purification tray: a. Pipette a 500-µL aliquot of the blood lysate into each well required. b. Operate the vacuum at 80% for 180 sec. c. Repeat steps a and b until all lysate is added. Note Up to six 500-µL aliquots of blood lysate may be added to each well, for a total of 750 µl of whole blood (equivalent to 3000 µl of blood lysate). Run the Method 5 If you are performing the... standard RNA isolation protocol ultra-low gdna protocol Then... a. Ensure that the highlighter is at step 1 of the RNA Blood method, then press F1 (Start). b. Perform run steps 1 to 9, as described under RNA Blood Method Steps on page 42. a. Ensure that the highlighter is at step 1 of the RNA Blood-DNA method, then press F1 (Start). b. Perform run steps 1 to 12, as described under RNA Blood-DNA Method Steps on page

46 RNA Blood Method Steps The RNA Blood method may be used for isolation of total RNA from whole blood or isolated blood cells. The steps required for this method are shown in the table below. IMPORTANT When you load the samples in step 1, multiple aliquots of lysate may be loaded to the purification tray well if necessary. However, take care to ensure that the purification tray membrane does not become overloaded and prevent the flow of wash solutions. A range of 5 to 750 µl of whole blood may be added to each purification tray well, equivalent to 20 to 3000 µl of lysate. For lysate volumes in excess of 650 µl, use the Quick Run feature and add lysate in 500 µl aliquots. Operate the vacuum and repeat until all of the lysate is added. Step Description Volume (µl) Position Time (sec) Vacuum (%) Prewet all wells with Wash 40 Waste Solution 1 1 Load samples 10 to 650 Waste Add Wash Solution Waste Add Wash Solution Waste Add Wash Solution Waste Add Wash Solution Waste Perform pre-elution vacuum Waste Touchoff at Waste Touch Off 8 Add Elution Solution 150 Collection Touchoff at Collection Touch Off 42

47 RNA Blood-DNA Method Steps The RNA Blood-DNA method may be used for isolation of total RNA from whole blood or isolated blood cells. It includes the removal of genomic DNA using AbsoluteRNA Wash Solution. The steps required for this method are shown in the table below. IMPORTANT When you load the samples in step 1, multiple aliquots of lysate may be loaded to the purification tray well if necessary. However, take care to ensure that the purification tray membrane does not become overloaded and prevent the flow of wash solutions. A range of 5 to 750 µl of whole blood may be added to each purification tray well, equivalent to 20 to 3000 µl of lysate. For lysate volumes in excess of 650 µl, use the Quick Run feature and add lysate in 500 µl aliquots. Operate the vacuum and repeat until all of the lysate is added.! CAUTION Do not operate the 6100 vacuum after the addition of AbsoluteRNA Wash Solution until the incubation following the addition of Wash Solution 2 has been completed in step 5. Operation of the vacuum before this time will remove the reagent from contact with the purification tray and increase the amount of genomic DNA present in the RNA sample. Step Description Volume (µl) Position Time (sec) Vacuum (%) Prewet all wells with Wash Solution 1 40 Waste 1 Load samples 10 to 650 Waste Add Wash Solution Waste Add Wash Solution Waste Add AbsoluteRNA Wash Solution and incubate 5 Add Wash Solution 2 and incubate 50 Waste See Caution above 400 Waste See Caution above 6 Remove Wash Solution 2 Waste Add Wash Solution Waste Add Wash Solution Waste Perform pre-elution vacuum Waste Touchoff at Waste Touch Off 11 Add Elution Solution 150 Collection Touchoff at Collection Touch Off 43

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