Standard Operating Procedure (SOP) Metabolomics

Transcription

1 Standard Operating Prcedure (SOP) Metablmics General Remarks Our platfrms perfrm targeted and nn-targeted metablmics analysis frm variety bilgical matrices, i.e. plasma, serum, tissues, urine, saliva, cells and cnditined cell medium f human and animal samples. T assure high quality results the fllwing guidelines need t be fllwed. Imprtant! Tube-Labels Label every single tube with prject name, sample number etc. Please be aware that the label-layer must nt exceed paper thickness. Otherwise the tubes might nt fit int ur rbt racks. Make sure that labelling is waterprf and resistant t cld strage cnditins. Stre the vials in labelled bxes (n bags!), rdered by sample number (crrespnding t yur sample list). Please fllw the guidelines - be aware that a surcharge (10 per sample) has t be claimed if labels r sample rder is nt crrespnding t this SOP. Please keep yur prcessing prcedures and times standardised and use same kind f bld cllectin and strage tubes fr all the samples f ne study t assure cmparability f results. Avid thawing f frzen samples: always use dry ice during handling and transprtatin. Cntent: - Plasma cllectin - Serum cllectin - Tissue cllectin - Sample transfer t HMGU - Prject descriptin frm - Recmmended tubes SOP Sample Preparatin fr Metablmic Platfrm versin 25/03/

2 Plasma cllectin and handling The preferred anticagulant fr plasma preparatin is EDTA but als heparin is acceptable. It is nt recmmended t use citrate! Ntes: - Fr targeted metablmics, amng ther methds, the Bicrates AbsluteIDQ kit is used. The Bicrates Kit is certificated fr human EDTA-Plasma samples. - The test persn/animal shuld be fasted fr eight hurs if pssible, unless a specific study designs is applied. 1) Cllect bld samples frm a peripheral vein directly int tubes*. Shake the tubes gently, but thrughly after finishing bld cllectin. D nt cl bld befre plasma separatin is finished. 2) Separate cells and plasma using centrifugatin as sn as pssible. Time frm bld cllectin t centrifugatin must nt exceed 40 min. Spinning-cnditins are as fllws: C, 10 min at 4500 x g (muse bld) r 2750 x g (human bld). 3) Transfer the plasma int a pre-cled cllectin tube (e.g. Falcn) withut aspirating bld cells; use dispsable pipette tips. Vrtex plasma and place n ice. 4) Label sample strage vials* (fr labelling see general remarks, page 1). Cl sample strage vials* and perfrm pipetting steps n ice. 5) Aliqut the plasma in suitable prtins int the labelled sample strage vials* t avid later freeze/thaw cycles. The filling f the vials must nt exceed three-quarters f their capacity. The minimal filling is depending n the vials** used. 6) Plasma samples need t be frzen immediately in liquid nitrgen and stred at -80 C. Stre the vials in bxes (n bags!), rdered by sample number. Ones frzen, samples must nt thaw Handle and transprt n dry ice! (If samples need t be thawed in rder t divide already frzen samples int smaller aliquts, samples shuld be thawed n ice, fllwed by prmpt redistributin int smaller aliquts and prmpt freezing) * Recmmended tubes fr bld taking and strage vials: see last page SOP Sample Preparatin fr Metablmic Platfrm versin 25/03/

3 Serum cllectin and handling (human) 1) Cllect bld samples frm a peripheral vein int the serum cllectin tube* with cltting activatr. 2) After finishing bld cllectin, shake the vial gently, but thrughly. 3) Stre the vial at rm temperature (20-28 C) in an upright psitin t allw cagulatin. Clt frmatin shuld be cmpleted after min in mst cases. If centrifugatin is nt perfrmed at the place f sample cllectin, use this time fr transprtatin. Time at rm temperature until centrifugatin shuld nt exceed 40 min. 4) Centrifuge t separate the serum frm the bld clt (15 C, 10 min, 4500 x g (muse bld) r 2750 x g (human bld)). 5) Transfer the serum int a pre-cled cllectin vial (e. g. Falcn) withut aspirating bld cells. Use dispsable pipette tips; Vrtex Serum and place n ice. 6) Label sample strage vials* (fr labeling see general remarks, page 1). Cl sample strage vials* and perfrm pipetting steps n ice. 7) Aliqut the serum in suitable prtins int the pre-cled, labeled strage vials* t avid later freeze/thaw cycles. The filling shuld nt exceed three-quarter f tubes capacity. The minimal vlume is depending n the vials* used. 8) Serum samples need t be frzen immediately in liquid nitrgen and stred at -80 C. Stre the vials in bxes (n bags!), rdered by sample number. Ones frzen, samples must nt thaw Handle and transprt n dry ice! (If samples need t be thawed in rder t divide already frzen samples int smaller aliquts, samples shuld be thawed n ice, fllwed by prmpt redistributin int smaller aliquts and prmpt freezing) * Recmmended tubes fr bld taking and strage vials: see last page SOP Sample Preparatin fr Metablmic Platfrm versin 25/03/

4 Tissue cllectin (animal) 1) Organ shuld be dissected as fast as pssible. Bld cntact with rgan surface has t be minimized. 2) If desired, rgan can be washed with istnic buffer slutin. 3) Pat tissue carefully with lint free tissue paper. 4) Cut Organs int apprpriate pieces f abut mg. Be careful t cllect always cmparable rgan-pieces f equal size (maximum 6 mm in diameter) frm the different animals! 5) Place samples int pre-cled, labelled tubes (fr labelling see general remarks, page 1). D nt add any slvents! 6) Tissue samples must be snap-frzen in liquid nitrgen and stred at -80 C until analysis. Stre samples in bxes (n bags!), rdered by sample number. Ones frzen, samples must nt thaw Handle and transprt n dry ice! Ntes: a. Targeted metablmics analysis: Different extractins can be perfrmed with tissue samples: Please cntact Crnelia Prehn fr discussing the aims f yur study. In sme cases, it can be beneficial t d tw different extractins. Please prepare ne tube per assay/extractin desired. Be careful t treat all samples in the same way. b. Nn-targeted metablmics analysis: Frzen r lyphilized tissue samples will be hmgenized with water (5 µl water/ mg tissue). The hmgenate will then be extracted with methanl. SOP Sample Preparatin fr Metablmic Platfrm versin 25/03/

5 Sample transfer Samples must nt thaw during transprtatin use dry ice in insulated bxes. Samples must be srted in bxes***, d nt use plastic bags. Each bx must be labelled n the side by prject name, respnsible persn, date f sample transfer (mnth/year), and bx number. Befre sending samples, first cntact respnsible staff t assure that ur lab is able t receive the parcel. Delivery t ur lab is nly pssible during wrking days. Ensure that the samples are dispatched t reach ur lab during wrking days (latest sending n Tuesday). T assure equal cnditins (cling etc.), samples f ne study shuld nt be send separately but tgether at ne time in ne parcel. Please enclse a printed versin f yur sample ID-list and the prject descriptin t the parcel. The same dcuments shuld be sent by (nly in XLS-frmat). Please prepare the sample list accrding t the prvided ExampleSampleList_Metablmics.xlsx. file: ExampleSampleList_Metablmics.xlsx SOP Sample Preparatin fr Metablmic Platfrm versin 25/03/

6 Cntact Persns: Dr. Crnelia Prehn phne: Dr. Anna Artati phne: Julia Scarpa prject crdinatin phne: fax: Deliver samples t: Julia Scarpa Helmhltz Zentrum München, German Research Centre fr Envirnmental Health (GmbH) Metablmic Platfrm, GAC, Institute f Experimental Genetics Building 34, Rm 336 Inglstaedter Landstrasse Neuherberg/Munich Germany SOP Sample Preparatin fr Metablmic Platfrm versin 25/03/

7 * Recmmended tubes fr bld taking Plasma: S-Mnvette 2.7 ml Kalium-EDTA, cde red, fr plasma separatin, with ptassium-edta, SARSTEDT AG & C., Nümbrecht, Germany, Art.-N (.001) (Alternatively, bld can be drawn with a pre-cled plastic syringe and subsequently transferred int a pre-cled EDTA/Heparin cated tube.. E. g. Prbenröhrchen zur Plasmagewinnung 1 ml, Lithium-Heparin, KABE Labrtechnik GmbH, Nümbrecht-Elsenrth, Art.-N. LI 1000 A Standrand r Prbenröhrchen für hämatlgische Untersuchungen, 1 ml, EDTA-di-Kaliumsalz, KABE Labrtechnik GmbH, Nümbrecht-Elsenrth, Atr.-N. EDTA 1000 A Standrand).In this case wrk quick but withut prducing fam n the bld surface Serum: S-Mnvette 2.7 ml Z, cde white, fr serum separatin, with additive carrier/clt activatr, SARSTEDT AG & C., Nümbrecht, Germany, Art.-N (.001) ** Sample strage vials: These vials have t be used manufacturer item Item n. min. sample vlume Eppendrf Safe-Lck-Vials 1,5 ml µl Eppendrf Safe-Lck-Vials 2 ml µl Bizym Vial 1,5 ml, screw cap µl Therm /Matrix 2D barcde tubes ml 50 µl If ther vial types shuld be used, please cntact staff befre preparing samples! *** Sample strage bx Freezer strage bxes shuld nt exceed 130 x 130 x 50 mm SOP Sample Preparatin fr Metablmic Platfrm versin 25/03/