SOP 25 Short Tandem Repeat (STR) Profiling

Transcription

1 Texas Cancer Cell Repsitry Cell Culture and Xengraft Repsitry SOP 25 Shrt Tandem Repeat (STR) Prfiling DNeasy Bld and Tissue Extractin: Cultured Cell Pellets 1) Begin with a thawed 2 x 10 6 cell pellet. Re-suspend in 200 µl f PBS (withut calcium and magnesium) and 20 µl prteinase K. 2) Add 200 µl AL buffer. Vrtex and incubate fr 10 minutes at 56 C. 3) Prepare and label spin clumns during incubatin. 4) Add 200 µl ethanl (100%) t the digested pellet. Vrtex thrughly. 5) Pipet the mixture int a 2 ml spin clumn (620 µl ttal). Centrifuge at 8000 rpm fr 1 minute. Discard waste and ld cllectin tube and replace with a new cllectin tube. 6) Add 500 µl AW1 buffer t spin clumn. Centrifuge at 8000 rpm fr 1 minute. Discard waste and ld cllectin tube and replace with a new cllectin tube. 7) Add 500 µl AW2 buffer t spin clumn. Centrifuge at 14,000 rpm fr 3 minutes. Discard waste and ld cllectin tube and replace with a labeled, sterile 1.5 ml r 2 ml micrcentrifuge tube. 8) Finally, add 200 µl f elutin buffer (AE r TE buffer) t the spin clumn. Incubate at rm temperature fr 1 minute. Centrifuge at 8000rpm fr 1 minute. 9) Quantify DNA yield using NanDrp (blank the NanDrp with the same liquid DNA is in) 260/280 rati shuld be between Stre DNA at -20 C fr shrt term and -80 C fr lng term Bld Begin with a 50 µl f separated RBCs. Add 170 µl f PBS (withut calcium and magnesium) and 20 µl prteinase K. Prceed with steps 2-9 abve. Tumrs Begin with a 5-25 mg tumr piece. Add 180 µl ATL buffer and 20 µl prteinase K. Incubate at 56 C until visible tumr is disslved (ccasinally vrtex). Add 200 µl AL buffer and 200 µl ethanl (100%). Vrtex thrughly. Prceed with steps 4-9 abve. Dilutins and Amplificatin Dilute DNA Prepare dilutins t 0.5ng/µL int 500 µl f sterile water. Prepare PCR strips All kit cmpnents are stred frzen and are returned t the freezer after use. Prmega GenePrint 10 System (part number B9510) reactin cmpnents: Prepare belw mixture int a sterile 1.5mL micrcentrifuge tube and vrtex. 5 µl per sample f Master Mix 5 µl per sample f Primer Pair Mix Add 10 µl f prepared Master Mix with Primer t each well f a PCR tube strip. Add 15 µl f each diluted sample (7.5ng f DNA fr each sample) Cntrl Tube: Add 1 µl f psitive DNA cntrl t 14 µl f amplificatin grade water with the 10 µl f Master Mix with Primer pairs Vrtex. Quick spin in the pst amp area and place in the thermal cycler. Date Revised: 11/18/2014

2 Amplificatin 25 µl Ttal Reactin Vlume Initial Incubatin Step 28 Cycles Final Extensin Final Hld 96 C 94 C 59 C 72 C 60 C 4 C 1 Min 10 Sec 1 Min 30 Sec 10 Min Capillary Electrphresis using the Applied Bisystems 3130xl Genetic Analyzer Set up Create a new plate under <plate manager> that crrespnds t the rientatin f the samples in the PCR strips. Each sample entry shuld read as fllws: Sample Name Results Grup: Results_Grup1 Instrument Prtcl: GP10 Machine Maintenance Add fresh 1x running buffer t the ande buffer reservir jar (16 ml) and cathde reservir tray, psitin 1 (16 ml). Add pure water t the tw water reservir trays, psitins 2 and 4, (16 ml each). Check fr bubbles in the plymer. Fr visible bubbles, fllw the bubble remve wizard Evaluate the POP-7 weekly 9at least 600 µl is required per run). Fr all ther maintenance, reference the Applied Bisystems Guide(s). Preparing Plate Cmbine 152 µl f HiDi Frmamide with 8 µl f ILS 600 size marker fr each grup f 16 wells. Vrtex. Never refreeze the HiDi after thawing Add 10 µl f prepared frmamide/size standard ccktail t each f the 16 wells (minimum). Add 1 µl f Allelic Ladder t the last well f each set f 16 wells. Add 1 µl f each amplified sample and each cntrl t the well that crrespnds t the prepared plate abve. Centrifuge the 96-well plate with septa fr secnds. Assemble the run plate. Place the plate assembly in the 3130xl Genetic Analyzer, clse the drs, <Link Plate> and <RUN> (green arrw). Reading Plates Samples are analyzed using GeneMapper ID v3.2.1 sftware Interpreting and Recrding Results Data Organizatin Data is rganized within an STR Access database by date and cell line name All samples typed (including repeats) are recrded int the STR Access database and rutinely updated int the COGcell.rg and TXCCR.rg STR database (this includes riginal patient material, cell lines, xengrafts, lymphblastid cell lines and fibrblast cell lines) Duplicate prfiles frm repeated testing f the same samples are recrded int the database t keep a histrical recrd f allelic patterns All prfiles are search-capable via the cell line name, cded patient identifier, date and the specific STR prfile The riginal data wrksheet fr each sample listing each lcus repeat number(s) and date f testing are kept alphabetically in binders after electrnic data entry. Verifying Knwn Prfiles TTUHSC Cancer Center/TXCCR/COGcell Shrt Tandem Repeat (STR) SOP Date Revised: 11/18/2014 Page 2 f 5

3 All cell lines are verified as a match against the riginal patient material All subsequently tested samples are then verified as an STR match when cmpared t previusly tested material within the STR Access database and COGcell.rg Any cmmercial lines are cmpared t available data thrugh the cell line prviders website (ex. ATCC r Sigma) All patient derived material shuld match the riginal prcessed samples Identifying Unique Samples Unique samples are thse that d nt have any riginal saved patient material t test against and d nt match any prfiles within the STR Access database, COGcell.rg/TXCCR.rg database, and available cmmercial search engines Fr these lines, clear ntes are made that the prfile is STR verified as unique, but that n riginal patient material is available Crss-Cntaminatin Fr samples that d nt match their riginal knwn prfiles within the STR Access database, COGcell.rg, and available cmmercial search engines all f the fllwing apply: Verify that the allele peaks had acceptable RFU values, if nt repeat the analysis. Verify that the crss-cntaminate is nt a labratry staff member. Cmpare the cntaminated prfile against ther lines the sample submitter is wrking with. If the cntaminant remains unknwn, discard all specimens assciated with that sample and g back t a cell line expansin passage and re-test that previus material befre cntinuing experiments Prfiles cntaining DNA frm mre than ne surce shuld be evaluated t determine pssible cntributrs. TTUHSC Cancer Center/TXCCR/COGcell Shrt Tandem Repeat (STR) SOP Date Revised: 11/18/2014 Page 3 f 5

4 Example f an STR Prfile within GeneMapperID The RFU are in the acceptable range (>200) Example f stutter; nt part f prfile Single-surce sample TTUHSC Cancer Center/TXCCR/COGcell Shrt Tandem Repeat (STR) SOP Date Revised: 11/18/2014 Page 4 f 5

5 GenePrint 10 Kit Lci Chrmsme Lcatin Amplicn Sizes THO1 11p bp D21S11 21q11-21q bp D5S818 5q bp D13S317 13q22-q bp D7S820 7q bp D16S539 16q24-qter bp CSF1PO 5q bp Amelgenin Xp and Y 106 and 112 bp vwa 12p bp TPOX 2p23-2pter bp TTUHSC Cancer Center/TXCCR/COGcell Shrt Tandem Repeat (STR) SOP Date Revised: 11/18/2014 Page 5 f 5