Application Protocol: Isolation of Genomic DNA from fresh and fixed rare cells

Transcription

1 REPRODUCTION AND USE This document is protected by copyright and it cannot be used or shared without permission from Vortex Biosciences, Inc. Such permission is given on condition that Vortex Biosciences is acknowledged whenever this protocol is reproduced or is published in whole or in part. PURPOSE This Application Protocol describes a method for isolation of genomic DNA from small amount of fresh, fresh frozen or fixed cells, such as Circulating Tumor Cells enriched with Vortex device from a human blood sample. This Protocol is using the QIAamp Micro kit from Qiagen with the following workflow, specifically designed for extraction of genomic DNA from small amounts of cells. MATERIALS Consumables Normal waste container. Chemical waste container. Paper towels. Pipette tips (p2, p10, p20, p200, p1000). 50 ml Falcon tubes. 10 ml serological pipette. Parafilm. Page 1 of 8

2 Maxymum Recovery Tubes (Microtubes MCT-150-L-C Axygen, 1.5 ml, ref # ) Plate sealing film (MicroAmp Optical Adhesive Film, Applied Biosystems, ref # ) Equipment Tube rack for eppendor tubes or falcon tubes. Pipettor (p2, p10, p20, p200, p1000). Vortexer. Minifuge. Bench top microcentrifuge with rotor for 2mL tubes, Eppendorf 5415 C or equivalent with maximum speed of g. Centrifuge with buckets for well plates, Beckman GS-6R or equivalent with maximum speed of 1000g.. Timer. Serological pipette aid. Thermomixer, heated orbital incubator, oven, heating block, bead bath or water bath with maximum temperature of 90 C. Rocker, if applicable (VWR mini rocker or equivalent). Plate sealing paddle. Relevant Personal Protective Equipment (PPE). Kits and Reagents (these should NOT be replaced without further validation) Virkon-S. Molecular biology grade Ethanol 70%. Molecular biology grade Ethanol 100%. UltraPure DNase, RNase free distilled water (Invitrogen, Cat# ). QIAamp DNA Micro kit (Qiagen, Cat#56304, store at RT.) Spin columns from the QIAamp DNA Micro kit from Qiagen (stored at 4 C). Carrier RNA prepared from the kit (aliquots stored at -20 C). PROCEDURE Preparing reagents (when starting from of an unopened kit) 1) Carrier RNA a) Prepare the Carrier RNA solution at 1 µg/ul by adding 310 µl of buffer AE to the tube containing 310 µg of lyophilized carrier RNA. b) Mix thoroughly until the lyophilized Carrier RNA is dissolved. Aliquot the solution by 30 µl or any other convenient size. c) Write down the product name, date of aliquoting and concentration. Store these aliquots at -20 C for up to 6 months. Do not freeze-thaw the aliquots of carrier RNA more than 3 times. Page 2 of 8

3 2) Buffer AW1 a) Add 25 ml of 100% ethanol to the bottle containing 19 ml of buffer AW1 concentrate. Tick the check box on the bottle label to indicate that ethanol has been added and write down the date. b) Mix the reconstituted buffer AW1 by shaking it and store at room temperature (15-25 C) for up to one year. 3) Buffer AW2 a) Add 30 ml of 100% ethanol to the bottle containing 13 ml of buffer AW2 concentrate. Tick the check box on the bottle label to indicate that ethanol has been added and write down the date. b) Mix the reconstituted buffer AW2 by shaking it and store at room temperature (15-25 C) for up to one year. Cell Preparation after collection with Vortex Technology, before or on the day of the DNA extraction Fresh cells/fresh frozen cells preparation a) Centrifuge the samples collected in microcentrifuge tubes or in a well plate at 1,000 g (2,000 rpm on the plate centrifuge or 3,500 rpm on the microcentrifuge) for 2 min. b) Carefully remove the supernatant without disturbing the cells at the bottom, leaving about 50 µl of solution in the tube or well. c) Proceed with the DNA extraction right away (fresh cells) or seal the plate tightly with parafilm and freeze the samples for later DNA extraction (fresh frozen cells). It is recommended to store the cells at -80 C for up to 6 months to prevent DNA degradation. Storage at -20 C is also possible for a short period like a month. Note: The DNA should be extracted as early as possible to avoid potential DNA degradation. Fixed cells preparation a) Fix the samples collected in microcentrifuge tubes or in a well plate by following Vortex Biosciences fixation protocol. b) If using a well plate, fill the wells with PBS to prevent evaporation and seal the plate tightly with parafilm. c) Store the samples at 4 C for up to one year. Setting up the experiment 4) Buffers and equipment a) Equilibrate the UltraPure water to room temperature (15-25 C). b) If buffers AL or ATL contain precipitates, dissolve them by heating the bottle(s) of solution at 70 C and mixing gently. c) Set a thermomixer or heated orbital incubator to 60 C for fixed cells or 56 C for fresh or fresh frozen cells. If thermomixers or heated orbital incubators are not available, heating blocks, oven, bead bath or water baths can be used instead. If the cells are stored in the well plate, use the heated orbital incubator or oven or bead bath to perform the incubation. d) Clean the workbench by spraying and wiping Virkon-S then 70% ethanol on the surface. Clean the pipette set with 70% ethanol. Page 3 of 8

4 Starting the experiment 5) Cell samples preparation Fresh cells: keep on ice. Fresh frozen cells: thaw the cell samples previously stored at -20 C or -80 C on ice and keep them on ice. Fixed cells: centrifuge the samples at 1,000 g (2,000 rpm on the plate centrifuge or 3,500 rpm on the microcentrifuge) for 2 min. Carefully remove the supernatant, leaving about 50 µl of solution in the tube or well. Keep on ice. Notes: - The supernatants can be transferred to low binding tubes such as maxymum recovery tubes and kept at 4 C until the DNA extraction has been performed and the DNA has been quantified. - From this point on, perform all centrifugation steps at room temperature (15-25 C). 6) Lysis Step a) Add 130 ul of buffer ATL to the samples. b) Add 20 µl of proteinase K. c) If using a well plate, mix the samples by pipetting up and down 10 times. Seal the plate using a sealing film and put back the cover on the plate. If using a tube, close the cap and mix by pulse-vortexing for 15 s. d) Incubate at 56 C for 20 min for fresh/frozen cells, or at 60 C overnight (maximum 18 hours) for fixed cells. Note: DNA yield of fixed cells can be increased by shaking the samples during the incubation. The use of a thermomixer or a heated orbital incubator is recommended. If not available, shake the well plate with a rocker at 300 rpm for 10 min or vortex the tubes several times during the incubation. e) During the incubation with buffer ATL/proteinase K, prepare the buffer AL/carrier RNA master mix: for each sample, mix 199 µl of buffer AL and 1 µl of dissolved carrier DNA. Multiply these volumes by the number of samples that need to be processed, and add 2 extra samples as a safety volume. Mix by inverting the tube at least 10 times. Do not Vortex since buffer AL contains SDS and thus makes lots of bubbles. Note: Adding carrier RNA to Buffer AL is always recommended for DNA extraction of very small amount of cells, such as circulating tumor cells isolated from blood. f) After the incubation, and if using a well plate, briefly centrifuge the plate at 200 g (1000 rpm) for a few seconds to bring all the liquid at the bottom of the wells. Mix each sample by pipetting up and down and transfer them to maxymum recovery tubes. Use one tube per well. g) Add 200 µl of buffer AL/carrier RNA master mix to each tube. Close the lid and mix thoroughly by pulse-vortexing for 15 s. Note: White precipitates may appear. Most of them will be dissolved after extended vortexing. If there are still some precipitates after vortexing, they should be dissolved after adding ethanol at step i). h) Briefly centrifuge the tubes to remove drops from inside of the lid. i) Add 200 µl of 100% ethanol to each tube. Close the lid and mix thoroughly by pulse-vortexing for 15 s. Note: If room temperature exceeds 25 C, cool the ethanol on ice before adding it to the tube. j) Incubate 5 min at room temperature (15-25 C). k) Briefly centrifuge the tubes to remove drops from inside of the lid. Page 4 of 8

5 7) Binding Step a) Carefully transfer each sample into a QIAamp MinElute column placed in a collection tube (without wetting the rim). Load maximum 700 µl per column. If several wells come from the same patient sample, combine the lysates onto the same column, 700 µl at a time. b) Close the lid and centrifuge at 6,000 g (8,000 rpm) for 1 min. c) Place each QIAamp MinElute column in a new, clean collection tube and discard the collection tube containing the flow-through. Collect the waste in a specific Guanidine waste container. d) If several wells come from the same patient sample, repeat steps a) through c) until all the lysate has been processed. 8) Washing Step a) Carefully open each QIAamp MinElute column and add 500 µl of buffer AW1. b) Close the lid, and centrifuge at 6,000 g (8,000 rpm) for 1 min. c) Place the QIAamp MinElute column in a new, clean collection tube, and discard the collection tube containing the flow-through. Collect the waste in a specific Guanidine waste container. d) Carefully open each QIAamp MinElute column and add 500 µl of buffer AW2. Close the lid, and centrifuge at 6,000 g (8,000 rpm) for 1 min. e) Place each QIAamp MinElute column in a clean collection tube, and discard the collection tube containing the flowthrough. f) Centrifuge at full speed (13,200 rpm for the benchtop microcentrifuge) for 3 min to dry the membrane. g) Label clean 1.5 ml maxymum recovery tubes with the samples ID, the date and the operator initials for elution. h) Place each QIAamp MinElute column in its corresponding elution tube and discard the collection tube containing the flow-through. 9) Elution Step a) Carefully open the lid of each QIAamp MinElute column and add µl of UltraPure water to the center of the membrane (expect a 5 µl loss through the filter during elution). Notes: - For downstream application that requires a small starting volume (such as multiplex PCR for GeneRead DNAseq panel assay), use an elution volume of 25 µl to increase the eluate concentration and the assay sensitivity. - Ensure that the ph of the water is at least 7.0. b) Close the lid and incubate at room temperature (15-25 C) for 5 min. c) Centrifuge at full speed (13,200 rpm for the benchtop microcentrifuge) for 1 min. d) Discard the column. e) Store the tubes containing the eluates at -20 C for later experiment or proceed directly with the DNA quantification by qpcr or Qubit. Page 5 of 8

6 Clean up Bring all equipment and supplies back. Spray & wipe down all surfaces with Virkon-S and 70% ethanol. Keep the shared space clean for next users. Throw away the well plate after the experiment is done. TROUBLESHOOTING Problem Cause Solution Little or no DNA in the eluate. Carrier RNA was not added to Buffer AL. Dissolve carrier RNA in Buffer AE [refer to section 1) Carrier RNA ] and mix the dissolved carrier DNA with Buffer AL as described in section 6) Lysis step, step e). Repeat the DNA extraction with new samples. Samples were frozen and thawed more than once. Avoid repeated freezing and thawing of samples. When possible, always use fresh samples or samples that have been thawed only once. Samples were kept at room temperature for too long. DNA in the samples may degrade during prolonged storage at room temperature. When possible, always use fresh samples, or samples stored at -20 C or -80 C. Insufficient sample lysis. Proteinase K was stored at high temperature for a prolonged time. Repeat the procedure using new samples and fresh proteinase K. Buffer AL/carrier RNA mixture was mixed insufficiently. Mix Buffer AL with carrier RNA by gently inverting the tube at least 10 times. Low-percentage of ethanol was used instead of %. Repeat the DNA extraction procedure with new samples and % ethanol. Buffer AW1 or AW2 was prepared incorrectly. Check that the Buffer AW1 and Buffer AW2 concentrates were diluted with the correct volume of % ethanol. Repeat the DNA extraction with new samples, if available. ph of water used for elution was too low. DNA does not dissolve easily in acidic solutions. Ensure that the ph of the water used for elution is >7.0. Page 6 of 8

7 IMPORTANT NOTES 1. Avoid contamination of samples and cross-contamination Proper microbiological aseptic techniques should always be used when working with small sample sizes. Hands and dust particles may carry bacteria and molds, and are the most common sources of contamination. Always wear lab coat and latex, nitrile or vinyl gloves. Change gloves frequently. When possible, avoid opening tubes by hand. It is recommended to use scissors or tweezers previously disinfected with 70% ethanol. Do not use the same pipette tip for different tubes. Be careful when working with tubes close to each other. Process one sample at a time and close each tube right after it is done being processed, before starting with another sample. 2. Keep track: Write down the date, the sample ID, the procedure and the operator s initials on both the top and side of the tubes. It is recommended to keep track of the sample processing in a database and update it at the end of each DNA extraction round. DOWNSTREAM ANALYSIS For DNA quantification, refer to the application protocol DQ001 (DNA quantification by qpcr) or DQ002 (DNA quantification by Qubit ). Comparison of DNA extraction yield for different kits (DNA extracted from HCT116 colon cancer cell line). Page 7 of 8

8 REFERENCES For the collection and fixation of samples, refer to the application protocol PB002 (Processing of human blood samples). EN-QIAamp-DNA-Micro-Handbook from Qiagen. Page 8 of 8