Circulating DNA from Plasma
Contents
1. 10 MACHEREY NAGEL 04 2007 Rev 01 Circulating DNA from Plasma 4 Safety instructions risk and safety phrases The following components of the NucleoSpin Plasma XS kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard Hazard Risk Safety Contents Symbol Phrases Phrases BB guanidine x Xn Flammable Harmful by inhalation in R 10 S 7 13 16 thiocyanate contact with skin and if swallowed 20 21 22 ethanol WB ethanol F Highly flammable R11 S 7 16 Proteinase K Proteinase K x Xn Irritating to eyes respiratory system R S 22 24 lyophilized Xi Jand skin may cause sensitization by 36 37 38 26 36 37 inhalation 42 Risk Phrases R10 Flammable R11 Highly flammable R 20 21 22 Harmful by inhalation in contact with the skin and if swallowed R 36 37 38 Irritating to eyes respiratory system and skin R 42 May cause sensitization by inhalation Safety Phrases S7 Keep container tightly closed S13 Keep away from food drink and animal feedstuffs S 16 Keep away from sources of ignition No Smoking S 22 Do not breathe dust S 24 Avoid contact with the skin S 26 Avoid contact with the eyes S 36 37 Wear suitable protective clothing and gloves Label not necessary if quantity below 125 g or ml concerning 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 42 and TRGS 200 7 1 MACHEREY NAGEL 042. applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 0 2421 969 270 e mail TECH BIO mn net com Last updated 12 2006 Rev 02 22 MACHEREY NAGEL 04 2007 Rev 01
3. 2 3 Kit specifications 5 2 4 Handling of sample material 7 2 5 Elution procedures 7 2 6 Removal of residual traces of ethanol for highest sensitivity 8 2 7 Stability of isolated DNA 9 3 Storage conditions and preparation of working solutions 10 4 Safety instructions risk and safety phrases 11 5 Protocols 12 5 1 High Sensitivity protocol for the isolation of DNA from plasma 12 5 2 Rapid protocol for the isolation of DNA from plasma 15 6 Appendix 17 6 1 Troubleshooting 17 6 2 Ordering information 18 6 3 Literature 18 6 4 Product use restriction warranty 21 MACHEREY NAGEL 04 2007 Rev 01 3 Circulating DNA from Plasma 1 Kit contents NucleoSpin Plasma XS 250 preps 10 preps 50 preps 740900 10 740900 50 740900 250 Binding buffer BB 4 5 ml 22 ml 110 ml Wash buffer WB 10 ml 2x25 ml 250 ml Elution buffer 5 ml 5 ml 13 ml Proteinase K 6 mg 30 mg 2x75mg lyophilized Proteinase Buffer PB 0 8 ml 1 8 ml 8 ml NucleoSpin Plasma XS 10 50 250 columns plus collecting tubes 2 ml collecting tubes 20 100 500 User manual 1 1 1 Reagents and equipment to be supplied by the user Thermal heating block Centrifuge for microcentrifuge tubes Manual pipettors and disposable pipette tips Microcentrifuge tubes 1 5 ml For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 04 2007 Rev 01 Circulating DNA from Plasma 2 Product description 2 1 The
4. 2007 Rev 01 11 NucleoSpin Plasma XS 5 Protocols Equilibrate sample to room temperature 18 C 25 C and make sure that the sample is cleared from residual cells cell debris and particulate matter e g by centrifugation of the plasma sample for 3 min at 211 000 x g For the High Sensitivity procedure Set the thermal heating block to 75 C 90 C for final ethanol removal see section 2 6 for details 5 1 High Sensitivity protocol for the isolation of DNA from plasma The High Sensitivity procedure is recommended if highest DNA yield and concentration is required The Rapid procedure 5 2 is recommended if shortest preparation time is required Optional Spike addition Pipet an appropriate DNA spike into the lid of a 1 5 ml reaction tube An appropriate DNA spike can be e g 20 ul of a solution containing one or several DNA fragments of 50 1000 bp with a concentration of 1 ng ul per fragment for subsequent analysis of DNA recovery e g via Agilent s Bioanalyzer 2100 1 Prepare sample Add 240 ul plasma to a microcentrifuge tube not provided 240 pl plasma Less than 240 ul may be used Adopt the binding buffer volume accordingly see below 1a Optional Proteinase K treatment Add 20 ul Proteinase K to the plasma sample mix and incubate at 37 C for 10 min Optional Depending on the plasma sample and the PCR conditions 20 ul the proteinase treatment of the plasma sample provokes a Prote
5. 2007 Rev 01 7 Circulating DNA from Plasma 20 pl 10 pl decreasing elution volume Fig 1 Correlation between elution volume and DNA concentration NucleoSpin Plasma XS columns 2 6 Removal of residual traces of ethanol for highest sensitivity A reduction of the 20 ul standard elution volume will increase the concentration of residual ethanol in the eluate For a 20 ul elution volumes a heat incubation of the elution fraction incubate eluate with open lid for 8 min at 90 C is recommended if the eluate comprises more than 20 of the final PCR volume in order to avoid an inhibition of sensitive downstream reactions In this context please mind the remarks below a An Incubation of the elution fraction at higher temperatures will increase signal output in PCR This is of importance especially if the template represents more than 20 of the total PCR reaction volume e g more than 4 ul eluate used as template in a PCR reaction with a total volume of 20 ul The template may represent up to 40 of the total PCR reaction volume if the eluate is incubated at increased temperature as described b A volume of 20 ul used for elution will evaporate to 12 14 ul during a heat incubation for 8 min at 90 C If a higher final volume is required please increase the initial volume of elution buffer e g from 20 ul to 30 ul c An incubation of the elution fraction for 8 min at 90 C will denature DNA If non denatured DNA is requi
6. Circulating DNA from Plasma User manual NucleoSpin Plasma XS April 2007 Rev 01 MACHEREY NAGEL MN Protocol at a glance Rev 01 Circulating DNA from Plasma NucleoSpin Plasma XS XS High Sensitivity protocol Rapid protocol 0 Optional Spike addition Spike addition 1 Prepare Sample r Use up to 240 ul plasma Use up to 200 pl plasma 1a Optional Proteinase K treatment 2 Adjust DNA binding conditions 3 Mix sample Add 20 ul Proteinase K mix incubate at 37 C for 10 min Add 360 pl BB Invert tube 3x vortex 3 sec spin down briefly Add 300 pl BB Invert tube 3x vortex 3 sec spin down briefly 4 Bind DNA 30 sec 2 000 x g 5 sec 11 000 x g 30 sec 11 000 x g 5 Wash and dry silica 500 pl wash buffer WB 500 pl wash buffer WB membrane LI i 30 sec 30 sec 11 000 xg 11 000 xg Pil 250 pl wash buffer WB Pi 250 pl wash buffer WB 3 min 3 min 11 000 x g 11 000 x g 6 Elute DNA 20 ul Elution Buffer 20 ul Elution Buffer 30 sec 30 sec 11 000 x g 11 000 x g 7 Removal of residual ethanol 8 min 90 C MACHEREY NAGEL GmbH amp Co KG e Neumann Neander Str 6 8 e D 52355 D ren e Germany Tel 49 0 24 21 969 270 e Fax 49 0 24 21 969 279 e mail tech bio mn net com Circulating DNA from Plasma Table of contents 1 Kit contents 4 2 Product description 5 2 1 The basic principle 5 2 2 About this user manual 5
7. DNA in human blood plasma length measurements in patients with pancreatic cancer and healthy controls Pancreas 1998 Jul 17 1 89 97 Hanley R Rieger Christ KM Canes D Emara NR Shuber AP Boynton KA Libertino JA Summerhayes IC DNA integrity assay a plasma based screening tool for the detection of prostate cancer Clin Cancer Res 2006 Aug 1 12 15 4569 74 Hromadnikova Zejskova L Doucha J Codi D Quantification of fetal and total circulatory DNA in maternal plasma samples before and after size fractionation by agarose gel electrophoresis DNA Cell Biol 2006 Nov 25 11 635 40 Jiang WW Zahurak M Goldenberg D Milman Y Park HL Westra WH Koch W Sidransky D Califano J Increased plasma DNA integrity index in head and neck cancer patients Int J Cancer 2006 Dec 1 119 11 2673 6 Jung M Klotzek S Lewandowski M Fleischhacker M Jung K Changes in concentration of DNA in serum and plasma during storage of blood samples Clin Chem 2003 Jun 49 6 Pt 1 1028 9 Koide K Sekizawa A Iwasaki M Matsuoka R Honma S Farina A Saito H Okai T Fragmentation of cell free fetal DNA in plasma and urine of pregnant women Prenat Diagn 2005 Jul 25 7 604 7 Lam NY Rainer TH Chiu RW Lo YM EDTA is a better anticoagulant than heparin or citrate for delayed blood processing for plasma DNA analysis Clin Chem 2004 Jan 50 1 256 7 MACHEREY NAGEL 04 2007 Rev 01 19 Circulating DNA from Plasma Lazar L Nagy B Ban Z Nag
8. Man CY Chiu RW Woo KS Lo YM Plasma beta globin DNA as a prognostic marker in chest pain patients Clin Chim Acta 2006 Jun 368 1 2 110 3 Epub 2006 Feb 14 Rhodes A Wort SJ Thomas H Collinson P Bennett ED Plasma DNA concentration as a predictor of mortality and sepsis in critically ill patients Crit Care 2006 10 2 R60 Schmidt B Weickmann S Witt C Fleischhacker M Improved method for isolating cell free DNA Clin Chem 2005 Aug 51 8 1561 3 Sozzi G Roz L Conte D Mariani L Andriani F Verderio P Pastorino U Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays J Natl Cancer Inst 2005 Dec 21 97 24 1848 50 Wang M Block TM Steel L Brenner DE Su YH Preferential isolation of fragmented DNA enhances the detection of circulating mutated k ras DNA Clin Chem 2004 Jan 50 1 211 3 20 MACHEREY NAGEL 04 2007 Rev 01 Circulating DNA from Plasma 6 4 Product use restriction warranty NucleoSpin Plasma XS kit components were developed designed distributed and sold for RESEARCH PURPOSES ONLY They are suitable for IN VITRO USES only No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin Plasma XS kits for a specific application range as the perform
9. ance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct i
10. basic principle The NucleoSpin Plasma XS kit is designed for the efficient isolation of circulating DNA from human blood plasma Fragmented DNA as small as 50 1000 bp can be purified with high efficiency Due to a special funnel design the NucleoSpin Plasma XS columns allow very small elution volumes 5 30 ul which results in highly concentrated DNA The protocol follows state of the art bind wash elute procedures After mixing of a plasma sample with the binding buffer the mixture is applied to the NucleoSpin Plasma XS column Upon loading of the mixture DNA binds to a silica membrane Two subsequent washing steps efficiently remove contaminations and highly pure DNA is finally eluted with 5 30 ul of a slightly alkaline elution buffer of low ionic strength 5 mM Tris HCl pH 8 5 2 2 About this user manual The manual provides two procedures differing in the number of handling steps speed and performance The High Sensitivity procedure is recommended if highest DNA yield and concentration is required The Rapid procedure is recommended if shortest preparation time is required Experienced users of NucleoSpin Plasma XS may refer to the Protocol at a glance instead of this user manual The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure First time users are strongly advised to read this user manual 2 3 Kit specifications e The Nuc
11. e The content of cell free DNA in human plasma may vary over several orders of magnitude DNA contents from approximately 0 1 1000 ng DNA per ml of plasma have been reported see remarks in section 2 3 Low DNA yield Sample contains residual cell debris or cells The plasma sample may have contained residual cells or cell debris Make sure to use only clear plasma samples see remarks in section 2 4 Column clogging No increase of PCR Residual ethanol in eluate signal despite of an increased volume of eluate used as template in PCR e Please see the detailed description of removal of residual traces of ethanol in section 2 6 Silica abrasion from the membrane e Due to the typically low DNA content in plasma and the resulting low total amount of isolated DNA a DNA quantification via Azgo absorption measuerment is often hampered due to the low sensitivity of the absorption measuement When performing absorption measurements close to the detection limit of the photometer the measurement may be influenced by minor amounts of silica abrasion In order to prevent incorrect Azgo quantification of small DNA amounts centrifuge the eluate for 30 sec at gt 11 000 x g and take an aliquot for measuement without disturbing any sediment Alternatively use a silica abrasion insensitive DNA quantification method e g PicoGreen fluorecent dye Discrepancy between Aze0 quantification values and PCR quantification value
12. ifuge for an additional 60 sec at 11 000 x g Wash silica membrane 1 wash Add 500 ul wash buffer WB to the column Centrifuge at 11 000 x g for 30 sec Discard collecting tube with flow through and place column into new collecting tube provided MACHEREY NAGEL 04 2007 Rev 01 360 pI BB mix sample load lysate gt gt er 30 sec CO 2 000 x g 5 sec pn CD 11 000xg 500 pl WB 30 sec 11 000 x g 13 NucleoSpin Plasma XS 6 Wash 2 wash and dry silica membrane Add 250 ul wash buffer WB to the column Centrifuge at 250 pl WB 11 000 x g for 3 min Discard collecting tube with flow through and place column into a 1 5 ml microcentrifuge tube 3 min for elution not provided 11 000 xg 7 Elute DNA 20 ul Add 20 ul Elution Buffer to the column Centrifuge at Elution Buffer 11 000 x g for 30 sec Elution volume may be varied from approximately 5 30 ul For a i 30 sec correlation of elution volume DNA concentration and DNA CS 11 000 x g amount eluted from the column see section 2 5 s 8 Removal of residual ethanol Incubate elution fraction with open lid for 8 min at 90 C gt e See section 2 6 for further comments and alternative incubation times and temperatures for a removal of residual ethanol 14 MACHEREY NAGEL 04 2007 Rev 01 NucleoSpin Plasma XS 5 2 Rapid protocol for the isolation of DNA from plasma The rapid procedure represents a good compro
13. inase K increase of the PCR signal of 0 5 1 5 cycles i e the cycle threshold Ct value crossing point Cp value is reached 0 5 1 5 cycles earlier The proteinase treatment may however alter the ratio of high to low molecular weight DNA 12 MACHEREY NAGEL 04 2007 Rev 01 NucleoSpin Plasma XS Adjust DNA binding conditions Add 360 ul binding buffer BB If less than 240 ul plasma is used adjust the binding buffer volume accordingly A ratio of 1 1 5 v v for plasma and binding buffer has to be ensured Mix sample Invert the tube 3 x and vortex for 3 sec Centrifuge the tube briefly to clean the lid Make sure to invert the tube especially if working with a spike in the tube lid vortexing alone does not securely flush liquid from the lid into the tube Bind DNA For each sample load the mixture 600 pl to a NucleoSpin Plasma XS column placed in a 2 ml collecting tube Centrifuge at 2 000 x g for 30 sec increase centrifuge force to 11 000 x g for further 5 sec Discard collecting tube with flow through and place column into new collecting tube provided The maximal column volume is approx 600 ul Do not apply a higher volume in order to avoid spillage If larger plasma sample volumes have to be processed the loading step may be repeated Be aware of an increased risk of membrane clogging in case of multiple column loading steps If the solution has not completely passed the column centr
14. ions temperature time and shaking rate for ethanol removal can be read from the diagram an initial volume of 20 ul will evaporate to 12 14 ul during the described incubation 2 7 Stability of isolated DNA Due to the typically low DNA content in plasma and the resulting low total amount of isolated DNA its fragmentation and the absence of DNase inhibitors the elution buffer does NOT contain EDTA the eluates should be placed on ice for short term and frozen at 20 C for long term storage MACHEREY NAGEL 04 2007 Rev 01 9 Circulating DNA from Plasma 3 Storage conditions and preparation of working solutions Attention The binding buffer BB contains guanidine thiocyanate and ethanol Wear gloves and goggles All kit components can be stored at room temperature 20 25 C and are stable up to one year Before starting any NucleoSpin Plasma XS protocol prepare the following e Before first use of the kit add Proteinase Buffer PB to dissolve lyophilized proteinase K as indicated see bottle or table below Proteinase K is stable at 4 C for up to 6 months Storage at 20 C is recommended if the solution will not be used up during this period NucleoSpin Plasma XS 10 preps 50 preps 250 preps 740900 10 740900 50 740900 250 Proteinase K 2x 75 lyophilized ome AMY Nees add 260 ul add 1 35 ml add to each vial Proteinase Buffer Proteinase Buffer 3 35 ml Proteinase Buffer
15. l 2005 The content of DNA in plasma obviously depends on condition of the donor sampling and handling of the blood plasma preparation and DNA isolation method DNA quantification method and others Size of circulating DNA A good portion of the cell free DNA in plasma is resulting from apoptotic cells As a result a considerable percentage of this circulating nucleosomal DNA is known to be highly fragmented The degree of fragmentation and the proportion of fragmented DNA relative to high molecular weight DNA however depends on several parameters like origin of the DNA e g fetal tumor microbial DNA health of the blood donor blood sampling procedure and handling of the sample The performance of many downstream applications consequently depends on the efficient isolation even of smallest DNA fragments Chan ef al 2006 2005 2004 2003 Deligezer et al 2006 Giacona et al 1998 Hanley et al 2006 Hromadnikova et al 2006 Jiang et al 2006 Koide et al 2005 Li et al 2006 2005 2004 Wang et al 2004 6 MACHEREY NAGEL 04 2007 Rev 01 Circulating DNA from Plasma According to this the NucleoSpin Plasma XS purification system is designed for the efficient isolation of highly fragmented DNA in a range of 50 1000 bp Within this range fragments are recovered with a similar high efficiency 2 4 Handling of sample material Several publications indicate strong influence of blood sampling handling storage and
16. leoSpin Plasma XS kit is recommended for the isolation of fragmented cell free DNA from human EDTA plasma e The NucleoSpin Plasma XS kit is designed for high recovery especially of fragmented DNA in a range of 50 1000 bp Up to 240 ul of plasma can be used as sample material DNA yield strongly depends on the individual sample but is typically in the range of 0 1 to 100 ng DNA per ml of plasma e Elution can be performed with as little as 5 30 ul elution buffer DNA is ready to use for downstream applications like real time PCR or others MACHEREY NAGEL 04 2007 Rev 01 5 Circulating DNA from Plasma The preparation time is approximately 15 30 min for 6 12 plasma samples Table 1 Kit specifications at a glance NucleoSpin Plasma XS Sample size Up to 240 ul EDTA plasma Average yield Typically in a range of 0 1 100 ng DNA per ml plasma depending on the sample Depending on kind of patient samples yield can be much higher Elution volume 5 30 ul Time prep High Sensitivity procedure 22 27 min 6 preps Rapid procedure 15 20 min 6 preps Spin column type NucleoSpin XS columns DNA yield from human plasma DNA amounts from less than 0 1 ng DNA per ml of plasma up to several 100 ng DNA per ml of plasma have been reported Chiu et al 2006 Chun et al 2006 Fatouros et al 2006 Lazar et al 2006 Rainer et al 2006 Rhodes et al 2006 Schmidt et a
17. mise between DNA yield and concentration as well as ease and speed of nucleic acid extraction Optional Spike addition Pipet an appropriate DNA spike into the lid of a 1 5 ml reaction tube An appropriate DNA spike can be e g 20 ul of a solution containing one or several DNA fragments of 50 1000 bp with a concentration of 1 ng ul per fragment for subsequent analysis of DNA recovery e g via Agilent s Bioanalyzer 2100 1 Prepare sample Add 200 yl plasma to a microcentrifuge tube not provided 200 ul plasma Less than 200 ul may be used Adopt the binding buffer volume accordingly see below 2 Adjust DNA binding conditions Add 300 ul binding buffer BB 300 ul BB If less than 200 ul plasma is used adjust the binding buffer volume accordingly A ratio of 1 1 5 v v for plasma and binding buffer has to be ensured 3 Mix sample Invert the tube 3x and vortex for 3 sec Centrifuge the tube briefly to clean the lid mix sample Make sure to invert the tube especially if working with a spike in the tube lid vortexing alone does not securely flush liquid from the lid into the tube MACHEREY NAGEL 04 2007 Rev 01 15 NucleoSpin Plasma XS Bind DNA For each sample load the mixture 500 pl to a NucleoSpin Plasma XS column placed in a 2 ml collecting tube load lysate Centrifuge at 11 000 x g for 30 sec Discard collecting tube with flow through and place column into new collecting t
18. ndirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty MACHEREY NAGEL 04 2007 Rev 01 21 Circulating DNA from Plasma Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all
19. plasma of nasopharyngeal carcinoma and lymphoma patients Cancer Res 2003 May 1 63 9 2028 32 Chan KC Zhang J Hui AB Wong N Lau TK Leung TN Lo KW Huang DW Lo YM Size distributions of maternal and fetal DNA in maternal plasma Clin Chem 2004 Jan 50 1 88 92 18 MACHEREY NAGEL 04 2007 Rev 01 Circulating DNA from Plasma Chiu RW Lo YM Noninvasive prenatal diagnosis by analysis of fetal DNA in maternal plasma Methods Mol Biol 2006 336 101 9 Chiu TW Young R Chan LY Burd A Lo DY Plasma cell free DNA as an indicator of severity of injury in burn patients Clin Chem Lab Med 2006 44 1 13 7 Chun FK Muller Lange Friedrich MG Erbersdobler A Karakiewicz PI Graefen M Pantel K Huland H Schwarzenbach H Circulating tumour associated plasma DNA represents an independent and informative predictor of prostate cancer BJU Int 2006 Sep 98 3 544 8 Deligezer U Erten N Akisik EE Dalay N Circulating fragmented nucleosomal DNA and caspase 3 mRNA in patients with lymphoma and myeloma Exp Mol Pathol 2006 Feb 80 1 72 6 Epub 2005 Jun 15 Fatouros IG Destouni A Margonis K Jamurtas AZ Vrettou C Kouretas D Mastorakos G Mitrakou A Taxildaris K Kanavakis E Papassotiriou Cell free plasma DNA as a novel marker of aseptic inflammation severity related to exercise overtraining Clin Chem 2006 Sep 52 9 1820 4 Epub 2006 Jul 13 Giacona MB Ruben GC Iczkowski KA Roos TB Porter DM Sorenson GD Cell free
20. plasma preparation on DNA yield and DNA quality Page et al 2006 Sozzi et al 2005 Chan et al 2005 Lam et al 2004 Jung et al 2003 Therefore it is absolutely recommended to keep blood sampling procedure handling storage and plasma preparation method constant in order to achieve highest reproducibility Plasma can be isolated according to protocols described in literature e g Chiu and Lo 2006 Birch et al 2005 or according to the following recommendation Preparation of plasma from human EDTA blood 1 Centrifuge fresh blood sample for 10 min at 2 000 x g 2 Remove the plasma without disturbing sedimented cells 3 Freeze plasma at 20 C for storage upon DNA isolation 4 Thaw frozen plasma samples prior to DNA isolation and centrifuge for 3 min at 211 000 x g in order to remove residual cells cell debris and particulate matter Use the supernatant for DNA isolation 2 5 Elution procedures The recommended standard elution volume is 20 ul A reduction of the elution volume to 5 15 ul will increase DNA concentration the total DNA yield is decreased by this reduction however An increase of the elution volume to 30 ul or more will only slightly increase total DNA yield but will reduce DNA concentration Figure 1 page 8 gives you a graphic description of the correlation between elution volume and DNA concentration and will thus help you to find the optimized elution volume for your individual application MACHEREY NAGEL 04
21. red like for downstream applications other than The maximum percentage of template volume in a PCR reaction may vary depending on the robustness of the PCR system 40 template volume were tested using LightCycler PCR Roche with the DyNAmo Capillary SYBR Green qPCR kit Finnzymes 8 MACHEREY NAGEL 04 2007 Rev 01 Circulating DNA from Plasma PCR e g ligation cloning we recommend an incubation for a longer time at a temperature below 80 C as most of the DNA has a melting point above 80 C Suggestion incubate for 17 min at 75 C d The incubation of the eluate at higher temperatures may be adjusted according to Fig 2 The incubation times and conditions shown will reduce an initial elution volume of 20 ul to about 12 14 ul and will effectively remove traces of ethanol as described above e If the initial volume of elution buffer applied to the column is less than 20 ul heat incubation times should be reduced in order to avoid complete dryness 25 without shaking N oO a 700 rpm 1400 rpm Incubation time min 65 70 75 80 85 90 95 Incubation temperature C Fig 2 Removal of residual ethanol from the elution fraction by heat treatment In order to obtain highest PCR sensitivity a heat incubation of the eluate is recommended Heat incubation may be performed at temperatures of 70 90 C ina heat block with or without shaking Effective condit
22. s MACHEREY NAGEL 04 2007 Rev 01 17 Circulating DNA from Plasma Measurement not in the range of photometer detection limit e In order to obtain a significant Az60 280 ratio it is necessary that the initially measured A260 and Azgo values are significantly above the detection limit of the photometer used An Azgo value close to the background noise of the photometer will cause unexpeced A260 280 ratios Unexpected A260 280 ratio 6 2 Ordering information Product Cat No Pack of NucleoSpin Plasma XS 740900 10 10 preps NucleoSpin Plasma XS 740900 50 50 preps NucleoSpin Plasma XS 740900 250 250 preps NucleoSpin collecting tubes 2 ml 740600 1000 6 3 Literature Birch L English CA O Donoghue K Barigye O Fisk NM Keer JT Accurate and robust quantification of circulating fetal and total DNA in maternal plasma from 5 to 41 weeks of gestation Clin Chem 2005 Feb 51 2 312 20 Epub 2004 Dec 17 Chan KC Lo YM Clinical applications of plasma Epstein Barr virus DNA analysis and protocols for the quantitative analysis of the size of circulating Epstein Barr virus DNA Methods Mol Biol 2006 336 111 21 Chan KC Yeung SW Lui WB Rainer TH Lo YM Effects of preanalytical factors on the molecular size of cell free DNA in blood Clin Chem 2005 Apr 51 4 781 4 Epub 2005 Feb 11 Chan KC Zhang J Chan AT Lei KI Leung SF Chan LY Chow KC Lo YM Molecular characterization of circulating EBV DNA in the
23. ube provided Dn 30 sec The maximal column volume is approx 600 ul Do not apply a E gt 11 000 x g higher volume in order to avoid spillage If larger plasma sample volumes have to be processed the loading step may be repeated Be aware of an increased risk of membrane clogging in case of multiple column loading steps If the solution has not completely passed the column centrifuge for an additional 60 sec at 11 000 x g Wash silica membrane 1 wash 500 pl WB Add 500 ul wash buffer WB to the column Centrifuge at 11 000 x g for 30 sec Discard collecting tube with flow 30 sec through and place column into new collecting tube a 11 000 x g provided CO Wash 2 wash and dry silica membrane Add 250 ul wash buffer WB to the column Centrifuge at KASOWE 11 000 x g for 3 min Discard collecting tube with flow through and place column into a 1 5 ml microcentrifuge tube 3 min for elution not provided er 11 000 x g Elute DNA 20 ul Add 20 ul Elution Buffer to the column Centrifuge at Punon Buter 11 000 x g for 30 sec Elution volume may be varied from approximately 5 30 ul For a i 30 sec correlation of elution volume DNA concentration and DNA ad N 11 000 x g amount eluted from the column see section 2 5 MACHEREY NAGEL 04 2007 Rev 01 Circulating DNA from Plasma 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Low DNA content of the sample
24. y GR Papp Z Presence of cell free fetal DNA in plasma of women with ectopic pregnancies Clin Chem 2006 Aug 52 8 1599 601 Epub 2006 Jun 1 Li Y Di Naro E Vitucci A Zimmermann B Holzgreve W Hahn S Detection of paternally inherited fetal point mutations for beta thalassemia using size fractionated cell free DNA in maternal plasma JAMA 2005 Feb 16 293 7 843 9 Erratum in JAMA 2005 Apr 13 293 14 1728 Li Y Holzgreve W DI Naro E Vitucci A Hahn S Cell free DNA in maternal plasma is it all a question of size Ann N Y Acad Sci 2006 Sep 1075 81 7 Li Y Holzgreve W Page Christiaens GC Gille JJ Hahn S Improved prenatal detection of a fetal point mutation for achondroplasia by the use of size fractionated circulatory DNA in maternal plasma case report Prenat Diagn 2004 Nov 24 11 896 8 Li Y Wenzel F Holzgreve W Hahn S Genotyping fetal paternally inherited SNPs by MALDI TOF MS using cell free fetal DNA in maternal plasma influence of size fractionation Electrophoresis 2006 Oct 27 19 3889 96 Li Y Zimmermann B Rusterholz C Kang A Holzgreve W Hahn S Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms Clin Chem 2004 Jun 50 6 1002 11 Epub 2004 Apr 8 Page K Powles T Slade MJ DE Bella MT Walker RA Coombes RC Shaw JA The Importance of Careful Blood Processing in Isolation of Cell Free DNA Ann N Y Acad Sci 2006 Sep 1075 313 317 Rainer TH Lam NY
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