Application Protocol: Plasma DNA Extraction using QIAamp Circulating Nucleic Acid kit

Transcription

1 REPRODUCTION AND USE This document is protected by copyright and 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 The QIAamp Circulating Nucleic Acid extraction protocol describes a method of efficient purification of free-circulating nucleic acids (e.g. cfdna) from human plasma. It comprises 4 steps (lysis, binding, wash, elution) and is performed using QIAamp Mini columns and a vacuum manifold (Figure 1). The robust procedure helps to eliminate sample-to-sample cross-contamination and increases user safety when handling potentially infectious samples. Plasma samples can be either fresh or frozen (provided they have not been frozen and thawed more than once). Extension tubes and vacuum processing on the QIAvac 24 Plus enable starting sample volumes of up to 5 ml, while flexible elution volumes between 20 μl and 150 μl allow concentration of nucleic acid species that are present in low amounts in the sample material. Figure 1: Workflow of the plasma DNA extraction. From QIAamp Circulating Nucleic Acid Handbook. Page 1 of 9

2 MATERIALS Consumables Normal waste container. Guanidine chemical waste container. Paper towels. Ice bucket and wet ice. Filtered pipette tips (p20, p200, p1000). 15 ml conical tube (Falcon tubes or equivalent). 50 ml conical tube (Falcon tubes or equivalent). Serological pipettes (5mL, 10 ml). 1.5 ml Maxymum Recovery microcentrifuge tubes or equivalent (Axygen, ref# MCT-150-L-C). Equipment Tube rack. Pipettors (p20, p200, p1000). Vortexer. Water bath or bead bath or heating block capable of holding 50 ml conical tubes at 60 C. Heating block or equivalent at 56 C (capable of holding 2 ml collection tubes). Bench top microcentrifuge with rotor for 2mL tubes, Eppendorf 5415 C or equivalent. Vacuum manifold (e.g., the QIAvac 24 Plus Qiagen, cat. no ) BSL II Biosafety cabinet. Vacuum pump capable of producing a vacuum of 800 to 900 mbar (e.g., QIAGEN Vacuum Pump or Evac Waste System, EV310, Argos Technologies Inc. or equivalent). Pipet-aid. Forceps. Suitable Personal Protective Equipment (PPE), including lab coat, gloves and eye protection. Kits and Reagents Virkon-S or equivalent. Bleach. Ethanol 70%. Ethanol Absolute (200 Proof, Molecular Biology Grade, Fisher BioReagents, ref# BP2818) UltraPure Distilled Water (Invitrogen ref# ) or equivalent. Page 2 of 9

3 Phosphate-buffered saline (PBS). Isopropanol (Fisher chemical, ref# A416 ) QIAamp Circulating Nucleic Acid Kit (50) (Qiagen, ref# 55114) PROCEDURE 1) Reagent and Equipment Preparation a) Always wear PPE (lab coat and gloves made of latex, nitrile or vinyl). b) For the biosafety hood, turn on the UV for 15 minutes. Clean with Virkon-S first and then 70% ethanol before the experiment. c) QIAamp Mini columns should be stored at 2 8 C upon arrival. d) All buffers can be stored at room temperature (15 25 C) for up to one year. e) The proteinase K inside the kit is stable for up to 1 year after delivery when stored at room temperature (15 25 C). To prolong the lifetime of proteinase K, storage at 2 8 C is recommended. f) Buffer ACL, Buffer ACB, and Buffer ACW1 contain guanidine salts, which can form highly reactive compounds when combined with bleach. If liquid containing these buffers is spilt, clean with suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) bleach. Buffer ACB: Before use, add 200 ml isopropanol (100%) to 300 ml buffer ACB concentrate to obtain 500 ml Buffer ACB. Mix well after adding isopropanol. Buffer ACW1: Before use, add 25 ml ethanol (96 100%) to 19 ml buffer ACW1 concentrate to obtain 44 ml Buffer ACW1. Mix well after adding ethanol. Buffer ACW2: Before use, add 30 ml ethanol (96 100%) to 13 ml buffer ACW2 concentrate to obtain 43 ml Buffer ACW2. Mix well after adding ethanol. Adding carrier RNA to Buffer ACL: o Add 1550 μl Buffer AVE to the tube containing 310 μg lyophilized carrier RNA to obtain a solution of 0.2 μg/μl. Dissolve the carrier RNA thoroughly, divide it into conveniently sized aliquots (e.g 50 μl), and store it at 15 to 30 C. Note that carrier RNA does not dissolve in Buffer ACL. It must first be dissolved in Buffer AVE and then added to Buffer ACL. Do not freeze thaw aliquots of carrier RNA more than three times. Page 3 of 9

4 o When use the carrier for the experiment, use 1 μg (5 μl dissolved carrier RNA in Buffer ACL, 0.2 μg/μl) of carrier RNA per sample. When make Buffer ACL carrier RNA mixture, gently mix by inverting the tube for 10 times to avoid foaming. Do not vortex. 2) Set-up of the QIAvac 24 Plus Vacuum Manifold a) Due to the large volumes of solution that need to go through the columns, a vacuum manifold such as the QIAvac 24 Plus, a waste bottle and a vacuum pump need to be connected together (Figure 2, left) to be able to draw all the solution through the column. The disposable VacConnectors (Figure 2, right) avoid any cross-contamination between samples. The VacValves (Figure 2, right) help to ensure a steady flow rate, facilitating parallel processing of samples of different natures, volumes, or viscosities. Figure 2: Set-up the Vacuum Manifold and Vacuum. (Left) QIAvac 24 Plus, QIAvac Connecting System, and Vacuum Pump. From Qiagen, QIAvac 24 Plus Handbook. (Middle) Evac Waste Vacuum system (EV310, Argos Technologies Inc.). From: (Right) Setting-up the QIAvac 24 Plus with QIAamp Mini columns using VacValves, VacConnectors, and Tube Extenders. (1) QIAvac 24 Plus vacuum manifold. (2) Luer slot of the QIAvac. (3) VacValve. (4) VacConnector 24 Plus (closed with luer plug). (5) QIAamp Mini column. (6) Tube Extender. From Qiagen, QIAvac 24 Plus Handbook. Page 4 of 9

5 b) Connect the QIAvac 24 Plus to a vacuum source. If using the QIAvac Connecting System, connect the system to the manifold and vacuum source following the QIAvac 24 Plus Handbook (Figure 2, left). If the Evac Waste Vacuum system (EV310, Argos Technologies Inc. Figure 2, middle) is used, then the extra waste bottle is not needed. c) Insert a VacValve into each luer slot of the QIAvac 24 Plus that is to be used (Figure 2, right). Close unused luer slots with luer plugs or close the inserted VacValve. VacValves should be used if flow rates of samples differ significantly to ensure consistent vacuum. d) Insert a VacConnector into each VacValve (Figure 2, right). Perform this step directly before starting the purification to avoid exposure of VacConnectors to potential contaminants in the air. e) Place the QIAamp Mini columns into the VacConnectors on the manifold (Figure 2, right). Save the collection tube for later use. f) Insert a tube extender (20 ml) into each QIAamp Mini column (Figure 2, right). Make sure that the tube extender is firmly inserted into the QIAamp Mini column to avoid leakage of sample. g) For nucleic acid purification, follow the below instructions. Discard the VacConnectors appropriately after use. Leave the lid of the QIAamp Mini column open while applying vacuum. Switch off the vacuum between steps to ensure that a consistent, even vacuum is applied during processing. For faster vacuum release, a vacuum regulator should be used. Each VacValve can be closed individually when the sample is completely drawn through the spin column, allowing parallel processing of samples of different volumes or viscosities. h) Please label the tubes, tube extenders and the QIAamp Mini columns before use on the QIAvac 24 Plus vacuum system to avoid mix-up of samples. i) After processing samples, clean the QIAvac 24 Plus following the below procedure. Disconnect the vacuum manifold from the vacuum pump. Remove the VacValves and soak in 1% (w/v) bleach for 10 minutes and then rinse thoroughly using water. Wipe dry or allow to air-dry. Discard the liquid waste from the QIAvac 24 Plus manifold to the chemical waste collection bottle. Rinse the manifold using 1% (w/v) bleach and then rinse it using water. It is important that all waste and buffers are completely drained from the manifold as they contain chaotropic salts, which can react with sodium hypochlorite. This cleaning procedure may reduce the operating life of the QIAvac 24 Plus manifold, due to the aggressive nature of sodium hypochlorite. Air dry the manifold to make it ready for the next use. The QIAvac 24 Plus should be regularly cleaned to maintain optimum performance. The QIAvac 24 Plus must also be Page 5 of 9

6 decontaminated before removal from the laboratory. Perform the cleaning procedure after each use to avoid sample contamination. 3) DNA Extraction from 5 ml Plasma a) Things to do before starting Always wear PPE (lab coat and gloves made of latex, nitrile or vinyl). Equilibrate plasma samples to room temperature if thawing from -80⁰C. Bring the volumes to 5ml with PBS if it is less than 5 ml. Set up the QIAvac 24 Plus as described above. Heat a bead bath or water bath or heating block to 60 C for use with 50 ml centrifuge tubes. Heat a heating block to 56 C for use with 2 ml collection tubes. If the available heat block does not fit 2 ml collection tubes, it is recommended to use 1.5 ml centrifuge tubes with the columns instead of 2 ml collection tubes for this step. Ensure that Buffer ACB, Buffer ACW1, and Buffer ACW2 have been prepared. Add carrier RNA reconstituted in Buffer AVE to Buffer ACL according to the calculation. b) Pipet 500 μl QIAGEN Proteinase K into a 50 ml centrifuge tube. c) Add 5 ml of plasma sample to the tube. d) Add 4 ml Buffer ACL (containing 1.0 μg carrier RNA). Close the cap and mix by pulse-vortexing for 30 s. Make sure that a visible vortex forms in the tube. To ensure efficient lysis, it is essential that the sample and Buffer ACL are mixed thoroughly to yield a homogeneous solution. Note: Do not interrupt the procedure at this time. Proceed immediately to next step to start the lysis incubation. e) Incubate at 60 C for 30 min. f) Place the tube back on the lab bench and unscrew the cap. g) Add 9 ml Buffer ACB to the lysate in the tube. Close the cap and mix thoroughly by pulse-vortexing for s. h) Incubate the lysate Buffer ACB mixture in the tube for 5 min on ice. i) Insert the QIAamp Mini column into the VacConnector on the QIAvac 24 Plus. Insert a 20 ml tube extender into the open QIAamp Mini column. Make sure that the tube extender is firmly inserted into the QIAamp Mini column in order to avoid leakage of sample. Keep the collection tube for later dry spin step. Page 6 of 9

7 j) Carefully apply the lysate Buffer ACB mixture from step 7 into the tube extender of the QIAamp Mini column. Switch on the vacuum pump. When all lysates have been drawn through the columns completely, switch off the vacuum pump and release the pressure to 0 mbar. Carefully remove and discard the tube extender. Please note that large sample lysate volumes (about 20 ml when starting with 5 ml sample) may need up to 15 minutes to pass through the QIAamp Mini membrane by vacuum force. For fast and convenient release of the vacuum pressure, the Vacuum Regulator should be used (part of the QIAvac Connecting System). Note: To avoid cross-contamination, be careful not to move the tube extenders over neighboring QIAamp Mini Columns. k) Apply 600 μl Buffer ACW1 to the QIAamp Mini column. Leave the lid of the column open, and switch on the vacuum pump. After all of Buffer ACW1 has been drawn through the QIAamp Mini column, switch off the vacuum pump and release the pressure to 0 mbar. l) Apply 750 μl Buffer ACW2 to the QIAamp Mini column. Leave the lid of the column open, and switch on the vacuum pump. After all of Buffer ACW2 has been drawn through the QIAamp Mini column, switch off the vacuum pump and release the pressure to 0 mbar. m) Apply 750 μl of ethanol (96 100%) to the QIAamp Mini column. Leave the lid of the column open, and switch on the vacuum pump. After all of ethanol has been drawn through the spin column, switch off the vacuum pump and release the pressure to 0 mbar. n) Close the lid of the QIAamp Mini column. Remove it from the vacuum manifold, and discard the VacConnector. Place the QIAamp Mini column in a clean 2 ml collection tube, and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. o) Place the QIAamp Mini Column into a new 2 ml collection tube (from the original column package). Open the lid, and incubate the assembly at 56 C for 10 min to dry the membrane completely. p) Place the QIAamp Mini column in a clean 1.5 ml elution tube (provided) and discard the 2 ml collection tube from step 14. Carefully apply 50 μl of UltraPure distilled water to the center of the QIAamp Mini membrane for complete elution of bound DNA. Close the lid and incubate at room temperature for 5 min. Ensure that the water is equilibrated to room temperature (15 25 C). The recovered eluate volume will be up to 5 μl less than the elution volume applied to the QIAamp Mini column. q) Centrifuge in a microcentrifuge at full speed (20,000 x g; 14,000 rpm) for 1 min to elute the nucleic acids. r) The eluted DNA then can be used for the quantification and downstream EGFR assay. Page 7 of 9

8 TROUBLESHOOTING Problems Causes Solutions Little or no nucleic acids in the eluate Samples frozen and thawed more than once Inefficient sample lysis in Buffer ACL Buffer ACL carrier RNA mixture not sufficiently mixed Low-percentage ethanol used instead of % Buffer ACB prepared incorrectly Buffer ACW1 or Buffer ACW2 prepared incorrectly Buffer ACW1 or Buffer ACW2 prepared with 70% ethanol Repeated freezing and thawing should be avoided as this may lead to DNA degradation. Always use fresh samples or samples thawed only once. If QIAGEN Proteinase K was subjected to elevated temperature for a prolonged time, it can lose activity. Repeat the procedure using new samples and fresh QIAGEN Proteinase K. Mix Buffer ACL with carrier RNA by gently inverting the tube of Buffer ACL carrier RNA at least 10 times. Repeat the purification procedure with new samples and % ethanol. Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. Check that Buffer ACB concentrate was reconstituted with the correct volume of isopropanol Check that Buffer ACW1 and Buffer ACW2 concentrates were diluted with the correct volume of ethanol. Repeat the purification procedure with new samples. Check that Buffer ACW1 and Buffer ACW2 concentrates were diluted with % ethanol. Repeat the experiment with new samples. Page 8 of 9

9 DNA or RNA do not perform well in downstream enzymatic reactions Little or no DNA in the eluate Inappropriate elution volume used Buffers not mixed thoroughly See Little or no nucleic acids in the eluate above for possible reasons. Increase the amount of eluate added to the reaction if possible. Determine the maximum volume of eluate suitable for your amplification reaction. Reduce or increase the volume of eluate added to the amplification reaction accordingly. The elution volume can be adapted proportionally. Salt and ethanol components of wash Buffer ACW2 may have separated out after being left for a long period between runs. Always check for precipitates and mix buffers thoroughly before each run. Too much DNA, with a quantity way higher than the predicted DNA amount WBC contamination Use the right blood collection tubes When separating the plasma from the whole blood, follow the adequate procedures and make sure the plasma is free of WBCs. Two centrifugations are needed. We use LBGard tube for cfdna study. If EDTA tube is used, please make sure the plasma is separated within 4 hours. REFERENCES QIAamp Circulating Nucleic Acid Handbook QIAvac 24 Plus Handbook DQ001: DNA Quantification by qpcr MD001: EGFR mutation detection using ctegfr qpcr kit MD002: EGFR mutation detection in both CTCs and ctdna from a single tube of blood Page 9 of 9