PROTOCOL: REVERSE TRANSCRIPTION AND INDIRECT LABELLING OF TOTAL RNA FOR cDNA AND OLIGO ARRAYS ============================================================================================= COMMENTS ========= The samples to be compared are each labelled with a different fluorescent dye and then subjected to paired competitive hybridisations. Indirect labelling involves two steps, firstly, incorporation of amino-allyl dUTP by reverse transcription, and then attachment of the fluorescent dyes. The following is an adaptation of a protocol from the Ajioka group within the Department of Pathology, University of Cambridge. This protocol can be used to label as little as 3 ug of total RNA. However, the following is for up to 30 ug of total RNA. For each microarray slide you should have two target samples; one labeled with Cy3, the other with Cy5. Normally 10 ug of RNA should be used as a starting material. Microcon YM-30 column steps are approximate, and need to be optimized for your particular centrifuge. If you centrifuge for too long and the pellet is dry, reload waste and re-centrifuge. EQUIPMENT AND REAGENTS ====================== - dATP, dCTP, dTTP and dGTP (Sigma; Cat. No. dNTP-100A) - Amino-allyl dUTP (Sigma; Cat. No. A0410) - Oligohexamere - Nuclease free water - Cy3 and Cy5 mono-reactive dye pack, (Amersham; Cat. No. PA23001 and PA25001) - RNAsin (Promega; Cat. No. 18064-014) - Superscript III Reverse Transcriptase (Invitrogen; Cat. No.18080-044) - EDTA (BDH; Cat. No. 100935V) - Sodium hydroxide, (BDH; Cat. No. 102525P) - Tris-HCl solid (BDH; Cat. No. 443864E) - Sodium Carbonate buffer 0.2M (pH 8,5-9,0) - Microcon YM-30 concentrators (Millipore; Cat. No. 42410) - MinElute PCR Purification Kit (Qiagen; Cat. No. 28004) - Hydroxylamine (Sigma; Cat. No. H-2391) - DMSO, high purity - Sonicated Salmon Sperm DNA (Invitrogen; Cat. No. 15632-011) - Centrifuge - Hot-block - Speed Vac REMOVAL OF RNASE ================ All materials should be autoclaved and only handled using gloves to avoid RNase contamination. Glassware should be baked at 180 °C overnight. MilliQwater and solutions should be nuclease free. The work area can be cleaned using RNase Zap to further limit the risk of RNase contamination. If possible, keep a set of pipettes purely for RNA work. PROCEDURE ========= Preparation of 0.2 M Sodium Carbonate Buffer: pH 8.5 - 9.0. * Solution I: Dissolve 0.84g NaHCO3 in 50 mL DEPC H2O in a disposable sterile Falcon tube. * Solution II: Dissolve 1.05g Na2CO3, in 50 mL DEPC H2O in a disposable sterile Falcon tube. * Mix 45 mL of Solution I and 2.75 of mL Solution II in a disposable sterile Falcon tube. * Check the pH by aliquoting 5 mL into a new 15 mL Falcon tube, if needed adjust the pH by adding appropriate amount of Sol-I or Sol-II. Never check the pH directly in the stock solution as that is a common source of RNAase contamination. * Aliquot 0.2 mL into RNAase-free tubes, store at -20°C. Discard the tube after use or 24 hours, whichever comes first. Reagents to mix and aliquot: 1. Make a 50 x dNTP mix - 10 ul dATP (100 mM stock) - 10 ul dCTP (100 mM stock) - 10 ul dGTP (100 mM stock) - 4 ul dTTP (100 mM stock) - 6 ul amino-allyl dUTP (100 mM) Then mix together and add to the master mix as below. 2. Master Mix for one sample (total 14.6 ul) - 6 ul First strand buffer (comes with Superscript III) - 0.6 ul 50x dNTP mix made above - 3 ul 0.1 M DTT (provided with Superscript III) - 0.25 ul RNAsin - 2.75 ul RNase-free water - 2 ul Superscript III You can make a pre-mix without Superscript III and make aliquots before storing at -20 °C. Add Superscript III before use. Reverse transcription for amino-allyl dUTP incorporation: 3. RNA is isolated and purified using our standard protocol 4. Adjust the volume of RNA (normally 10 ug) to 12.5 ul with RNase-free water and add to a 1.5 ml RNase/DNase free microfuge tube 5. Add 3 ul of anchored Oligo hexamer primer to each RNA sample 6. Place the tubes at 65 °C for 10 minutes 7. Place the tubes immediately on ice for 5 minutes 8. Add 14.6 ul of the master mix to each tube (made above) and incubate at 42 °C for 2hrs 9. Remove the tubes from hot block, and add 10 ul 0.5 M EDTA and 10 ul of 1 M soduim hydroxide. Place at 65 °C for 15 minutes 10. Remove from heat and place at room temperature for 2 minutes 11. Add 25 ul of 1 M Tris-HCl (pH 7.5), mix 12. Add 450 ul of RNase-free water to each sample and add each sample to a microcon-YM30 concentrator. Add sample without touching the membrane. 13. Centrifuge at room temperature at 13,000 rpm for about 7 minutes (or until about 50 ul of water left in reservoir), empty waste, and then add another 400 ul of water. 14. Centrifuge again at room temperature at 13,000 rpm for 7 minutes 15. Empty waste and add 400 ul water for a third time 16. Centrifuge tube at room temperature at 13,000 rpm for 8 minutes (until volume left reached about 10 ul). 17. Invert the column into new 1.5 ml collection tube. 18. Centrifuge at RT at 3,000 rpm for 4 minutes. 19. Place the tubes in the speed vac to dry on medium heat. Can be stored at -20 °C. Dye attachment: 20. Resuspend the pellet in 4.5 ul 0.2 M Sodium Carbonate buffer (pH 8.5-9.0). 21. If necessary resuspend one aliquot of Cy3 or Cy5 dye in 36.5 ul of DMSO (sufficient for 8 labelling reactions), vortex and spin content down 22. Mix 4.5 ul of resuspended dye with resuspended pellet. 23. Place in the dark for 1 hour at 23 °C (in a hot block). Dye quenching and removal of unincorporated dye 24. Add 4.5 ul of 4 M hydroxylamine to each sample to stop reaction and incubate in the dark for 15 minutes at 23 °C. Clean up samples using Qiagen MinElute PCR Purification Kit: 25. Add 35 ul of 3 M sodium acetate (pH 5.2) to each reaction, and mix both samples (Cy3 and Cy5) in a new 1.5 ml microfuge tube 26. Add 5 volumes (500 ul) of buffer PB and mix. 27. Place a MinElute column in a provided 2 ml collection tube. 28. To bind DNA, apply the sample to the MinElute column and centrifuge for 1 minute at 13,000 rpm. 29. Discard flow through, and add 750 ul of Buffer PE to the column. 30. Centrifuge for 1 minute at 13,000 rpm. 31. Repeat steps 29 and 30. 32. Discard flow through and place MinElute column back in same tube and centrifuge empty column for 1 minute at 13,000 rpm. 33. Place column into a new 1.5 ml microfuge tube and add 10 ul elution buffer (nuclease free water). 34. Incubate at room temperature for 1 minute in dark. 35. Centrifuge for 1 minute at 13,000 rpm. 36. Add another 10 ul of RNase-free water to column and centrifuge for 1min at 13,000rpm. Collect in same tube. 37. Dry in the speed vac to a volume of 2 to 5 ul (about 10 minutes). 38. Add 2 ul sonicated salmon sperm DNA (from 10 mg / ml stock). The two samples (i.e. sample and control) have been combined together for hybridisation to a microarray and the blocking agent, sonicated salmon sperm DNA, has been added. This material should now be used immediately to prevent any decay. Please refer to the appropriate hybridisation protocol for the next steps. PROTOCOL VERSION ================ 1.0 / 14.12.2007 Author: Marisch, Karoline AUSTRIAN CENTER OF BIOPHARMACEUTICAL TECHNOLOGY
Labeled Extracts were pooled and mixed. The amount (ul) of each individual Labeled Extract used for pooling is described by protocol parameter [QuantityUsed].
Microarray Hybridisation Protocol ================================== EQUIPMENT AND CHEMICALS ======================= Albumin-Fraction V (BSA) [Applichem, A1391] M ~ 68000g/mol SDS 10% (Dodecylsulfate sodiumsalt) [Merck , #106022] SSC Buffer (20x) [Invitrogen, #15557-044] SSPE Buffer (20x) [Fluka, #85637] OpArray HybSolution [Operon, #] Custom Oparray [Operon] HQ-Wasser Eppendorf Thermomixer comfort Tecan HS400 REAGENTS TO PREPARE =================== All solutions are calculated for the use of all four slides in the TECAN HS400. Blocking buffer (400 ml): 4x SSC 80 ml 20x SSC 0.5% (v/v) SDS 20 ml SDS 10% 1% BSA 4g BSA HQ-H2O 300 ml Filtrate solution through a 0.22 um filter. Pre-hybridization buffer: 5x SSPE 50 ml 20x SSPE HQ-H2O 150 ml Washing buffer 1: 2x SSC 50 ml 20x SSC 0.1% SDS 5 ml SDS 10% HQ-H2O 445 ml Washing buffer 2: 1x SSC 25 ml 20x SSC HQ-H2O 475 ml Washing buffer 3: 0.5xSSC 12.5 ml 20x SSC HQ-H2O 487.5 ml PROCEDURE ========== Sample preparation: * Add OpArray HybSolution to each sample to obtain a final volume of 70 ul (volume of the hybridization chamber). Concentration of the HybSolution in the samples should be 90 %. * Heat samples for 3 min at 95°C on a hot-block. Preparation of Tecan HS400: Carefully read and follow all instructions shown on the screen and be very careful at sample injection steps. A + marks the chamber into which the sample should be injected and this has to be confirmed after each injection step on the station by pressing the OK button. * Open HS400 control application * Start a program * Connect the instrument * Put all tubes in the bottles. * Check numbers on tubes and bottles. * Prime the first used channel (5) to fill the tube * Switch on heating if necessary * Switch on the nitrogen supply * Open hybridization shell * Insert chambers into positions * Insert slides into slide positions, insert dummy slides into all unused positions * Close hybridization shell * Insert connectors * Start program and follow the instructions on the display carefully. * Select only chambers which are used during hybridization run. PROGRAM START 1.WASH: Temp. °C:42.0,First: Yes, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:30 2.HYBRIDIZATION: Temp. °C:42.0, Agitation Frequency: High, Time: 0:07:00 3.WASH: Temp. °C:42.0,First: No, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:30 4. HYBRIDIZATION: Temp. °C:42.0, Agitation Frequency: High, Time: 0:07:00 5.WASH: Temp. °C:42.0,First: No, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:30 6.HYBRIDIZATION: Temp. °C:42.0, Agitation Frequency: High, Time: 0:07:00 7.WASH: Temp. °C:42.0,First: No, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:30 8.HYBRIDIZATION: Temp. °C:42.0, Agitation Frequency: High, Time: 0:07:00 9.WASH: Temp. °C:42.0,First: No, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:30 10.HYBRIDIZATION: Temp. °C:42.0, Agitation Frequency: High, Time: 0:07:00 11.WASH: Temp. °C:42.0,First: No, CH.:6 Runs 6, Wash time: 0:03:00, Soak time: 0:01:00 12.WASH: Temp. °C:42.0,First: No, CH.:1 Runs 1, Wash time: 0:01:00, Soak time: 0:01:00 13.PROBE INJECTION: Temp. °C:50.0 -> 70ul hybridization buffer 14.HYBRIDIZATION: Temp. °C:50.0, Agitation Frequency: Medium, Time: 0:01:00 15.PROBE INJECTION: Temp. °C:50.0 -> 70ul sample 16.HYBRIDIZATION: Temp. °C:50.0, Agitation Frequency: Medium, Time: 16:00:00 17.WASH: Temp. °C:42.0,First: No, CH.:2, Runs 4, Wash time: 0:001:00, Soak time: 0:03:00 18.WASH: Temp. °C:42.0,First: No, CH.:3 Runs 1, Wash time: 0:01:00, Soak time: 0:03:00 19.WASH: Temp. °C:30.0,First: No, CH.:3 Runs 4, Wash time: 0:01:00, Soak time: 0:03:00 20.WASH: Temp. °C:30.0,First: No, CH.:4 Runs 4, Wash time: 0:01:00, Soak time: 0:03:00 21.SLIDE DRYING: Temp. °C:30.0, Time: 0:02:00, Final Manifold Cleaning: No, Ch.: No PROGRAM END Liquid identification: Liquid for channel 1: prehybridization buffer Liquid for channel 2: 2xSSC, 0.1% SDS Liquid for channel 3: 1xSSC Liquid for channel 4: 0.5xSSC Liquid for channel 5: blocking buffer Liquid for channel 6: HQ-H2O After Hybridization: After the slide hybridization a pop-up message appears (Run finished OK). Quit message and take out slides for scanning. * Insert dummy slides into all slide positions and close chamber shell * Start the sds-wash program to remove remaining contaminations and follow the instructions carefully PROGRAM START 1.WASH: Temp. °C: 40.0, First: YES; CH.: 5; Runs: 5, Wash time: 0:01:00, Soak time: 0:02:00 2.WASH: Temp. °C: 40.0, First: YES; CH.: 6; Runs: 5, Wash time: 0:02:00, Soak time: 0:02:00 3.SLIDE DRYING: Temp. °C:40.0, Time: 0:00:30, Final Manifold Cleaning: No, Ch.: No PROGRAM END Liquid identification: Liquid for channel 1: Liquid for channel 2: Liquid for channel 3: Liquid for channel 4: Liquid for channel 5: 0.1% SDS Liquid for channel 6: HQ-H2O * After the sds-wash program is finished the RINSE step should be run. * All tubes should be inserted into HQ-water * After the display message: remove all tubes from liquid this should be done and the final system drying step should be started * After finishing RINSE the chambers can be removed from the HS400. * Disconnect the instrument. * Switch of the heating if previously turned on. * Put chambers into HQ-water and incubate for about 1 h at 60°C. * Dry chambers with nitrogen and store them until next use. PROTOCOL VERSION ================ 1.0 / 07.01.2008 Author: Strobl, Florian AUSTRIAN CENTER OF BIOPHARMACEUTICAL TECHNOLOGY 2.0 / 26.05.2008 Author: Marisch, Karoline AUSTRIAN CENTER OF BIOPHARMACEUTICAL TECHNOLOGY
---------------------- limma Normalization ---------------------- Background Subtraction Method: none Within Slide Normalization Method: none Between Slide Normalization Method: vsn Use Dye Swap Pairs: yes Average Spots: yes ------------------------------------------------------------------------ ------------------------------------------------------------------------ R version 2.8.1 (2008-12-22) x86_64-pc-linux-gnu locale: LC_CTYPE=en_US;LC_NUMERIC=C;LC_TIME=en_US;LC_COLLATE=en_US;LC_MONETARY=C;LC_MESSAGES=en_US;LC_PAPER=en_US;LC_NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=en_US;LC_IDENTIFICATION=C attached base packages:  tools stats graphics grDevices datasets utils methods  base other attached packages:  vsn_3.8.0 affy_1.20.0 Biobase_2.2.0 lattice_0.16-2 limma_2.16.2 loaded via a namespace (and not attached):  affyio_1.10.1 grid_2.8.1 preprocessCore_1.4.0
RNA Extraction Protocol ======================= EQUIPMENT AND CHEMICALS ======================= Nuclease-free water [Ambion, #9932] EDTA (ethylene diamine tetraacetic acid) [Merck, #108418] M: 372.24 g/l NaOH pellets, extra pure [Merck; #106482] Lysozyme [Amresco, #0663-5G] Trizol [Invitrogen, #15596-018] Chloroform [Sigma-Aldrich; #C-2432] Isopropanol [Sigma-Aldrich; #I-9516] Tris [Roth; #5429.1] M: 121.14 g/l EtOH absolute [Sigma; #32221] M: 46.07 g/l Hydrochloric acid (32 %) [Merck, #100319] Eppendorf Centrifuge 5415R Scientific Industries Vortex Genie 2 NanoDrop ND1000 Eppendorf Thermomixer comfort REAGENTS TO PREPARE =================== All solutions should be prepared with nuclease-free water. Glass ware should be baked at 180°C over night. Always use gloves and RNase-free tips. Extraction steps must be carried out under the extractor hood. 0.5 M EDTA (pH= 8.0) Dissolve 18.6 g of EDTA in 80 ml nuclease-free H2O, adjust the pH to 8.0 with NaOH pellets and fill up to 100 ml with nuclease-free water. 1M Tris-HCl (pH= 7.4) Dissolve 60.57 g of Tris in 400 ml nuclease-free H2O, adjust the pH with 37% HCl to 7.4 and fill up to 500ml with nuclease-free water. TE-buffer (pH= 7.5) Mix 1 ml of 0.5M EDTA (pH= 8.0) and 5 ml 1M Tris-HCl (pH= 7.4) and fill up to 500 ml with nuclease-free water. Lysozyme solution (10 mg/ml) Dissolve 100 mg of lysozyme in 10 ml of TE-buffer. Aliquote per 500 ul in RNase-free reaction tubes. Ethanol (75 %) Mix 37.5 ml of EtOH absolute with 12.5 ml of nuclease-free water. PROCEDURE ========= This protocol can be used for samples taken during fermentations according to the sample taking protocol for RNA samples. Samples should contain 3 mg/mg BDM per aliquot. Samples should be stored at -80°C. The RNA-extraction procedure is adjusted to a 1.5 ml scale. - Unfreeze and vortex pellet (1 min on the vortex) - Add 100ul of lysozyme solution (10mg/ml in TE-buffer) - Vortex pellet for 5 min at room temperature to resuspend bdm - Add 800 ul of trizol and vortex - Incubate for 5 min at room temperature without vortexing - Add 160ul of chloroform and vortex for 1 min at room temperature - Incubate for 2 min 30 sec at room temperature - Centrifuge at 12.000 g (13400 rpm) for 15 min at room temperature - Transfer the upper aqueous phase (500 ul) to a new RNase free reaction tube - Add 2 volumes of isopropanol (1000 ul) - Incubate for 15 min at room temperature - Centrifuge at 12.000 g (13400 rpm) for 15 min at 4°C - Discard supernatant and wash pellet with 1 ml of 75% EtOH. (RNA can be stored at this step at -20°C until use.) - Centrifuge at 12.000 g (13400 rpm) for 15 min at 4 °C - Discard supernatant - Air-dry pellet (or at 37°C on a hot-block) and dissolve in 100 ul of nuclease-free water Concentration of isolated RNA is measured with the NanoDrop and the quality should be controlled with the Bioanalyzer according to the manufacturer’s protocols. PROTOCOL VERSION ================ 1.0 / 26.05.2008 Author: Marisch, Karoline AUSTRIAN CENTER OF BIOPHARMACEUTICAL TECHNOLOGY
CHEMICALS AND EQUIPMENT Phenol (Tris-HCl-saturated, pH= 6.7 ? 0.2) [Sigma P-4557] EtOH absolute [Sigma; #32221] M: 46.07 g/l Syringes sterile (10 ml) RNase-free reaction tubes 1.5 ml Eppendorf Centrifuge 5415R REAGENTS TO PREPARE Phenol/Ethanol stabilising solution (5%) Mix 2.5 ml of phenol with 47.5 ml of EtOH absolute (calculate required volume of solution for each cultivation and modify if necessary (2ml per sample taking)). PROCEDURE This protocol can be used for RNA-sample drawing in E. coli cultivations. Approximately 3 mg of biomass dry matter will be drawn of the bioreactor and immediately stabilised with the solution prepared above. Always use RNase-free tips and reaction tubes, wear nitril-gloves. All sample preparation steps should be carried out under the extractor hood. - Pipette 2 ml of phenol/ethanol stabilising solution into 10 ml syringe - Pull plunger to a point on the scale - Draw accurately 4 ml of fermentation broth into the syringe and shake immediately - Using the formula (1) calculate volume of suspension for aliquoting into cooled 1.5 ml RNase-free reaction tubes (use the theoretical biomass dry matter of the fermentation and prepare before; tubes should contain 3 mg of bdm) - Centrifuge samples at 13400 g and 4°C for 2 min - Discard supernatant - Store at -80°C. (1) REVISION Version generated on from modifications 1 23.03.2004 Striedner n.A. 2 26.05.2008 Marisch Translation to english AUSTRIAN CENTER OF BIOPHARMACEUTICAL TECHNOLOGY Author: StriednerHelga Date: 23.03.200422.12. Working group MO-Fermentation PROTOCOL: SAMPLE TAKING PROCEDURE OF RNA IN E. COLI CULTIVATIONS Version: 2 PAGE 1 OF 1 (c) MO-Ferm AG
Growth Protocol for Strain Comparison ====================================== REACTOR ======== 20 l MBR Bioreactor (14l working volume) PROCESS CONTROL ================ SPS: Siemens Simatic S7 (Siemens Automation) SCADA: customized process automation designed by ISE (Industrial Software Engineering GmbH, Vienna) based on Proficy HMI SCADA - iFIX 4.0 (GE Fanuc Intelligent Platforms Europe S.A., Darmstadt Germany) process data storage: Proficy iHistorian 3.0 ON-LINE MONITORING DEVICES AND CONTROL STRATEGIES ================================================== pH probe: Mettler Toledo, pH2100e, InFit 761e control: pH control by addition of 25 % ammonia solution (MERCK) setpoint: 7 +/- 0.05 pO2 probe: Mettler Toledo InPro?6800 control: control of by stirrer speed and aeration rate setpoint: dissolved oxygen level > 30% Temperature probe: Pt100 (Thermoest, Vienna, A) control: double jacket cooling/heating system setpoint: 37 °C +/- 0.05 CO2 and O2 offgas measurement Advanced Optima gas analyzer, Hartmann and Braun, ABB Austria Capacitance and conductivity measurement Aber Instrument model 214M (Aber Instruments, Aberystwyth, UK). Multi-wavelength fluorescence measurement BioView(R) multi-wavelength spectrofluorometer, DELTA Light & Optics, Lyngby, Denmark Measurement of volatile components in the offgas High sensitivity PTR-MS Ionimed Innsbruck, Austria MEDIUM ====== Minimal medium: 3 g KH2PO4 and 6 g K2HPO4.3H2O per liter (buffer capacity and also P and K sources). All other components added in relation to the gram cell dry weight to be produced: 0.25 g sodium citrate (trisodium salt.2H2O; ACROS organics), 0.10 g MgSO4.7H2O, 0.02 g CaCl2.2H2O, 50 ul trace element solution, and 3 g glucose.H2O. The trace element solution was prepared in 5 N HCl and contained (g/l) 40.0 FeSO4.7H2O, 10.0 MnSO4.H2O, 10.0 AlCl3.6H2O, 4.0 CoCl2 (Fluka), 2.0 ZnSO4.7H2O, 2.0 Na2MoO2.2H2O, 1.0 CuCl2.2H2O, and 0.50 H3BO3. CULTIVATION MODE ================ Batch The minimal medium for batch processes is supplemented with 0.15 g yeast extract per gram CDW to be produced to accelerate initial growth of the population. For inoculation, an overnight (o/n) culture, which was inoculated with 10 ul of a working cell-bank, is transferred aseptically to the bioreactor (CDW of o/n culture corresponded to ~ 0.38 g). Foaming was suppressed by addition of 0.5 ml antifoam suspension (Glanapon 2000, Bussetti, Vienna) per litre medium. Settings: Batchvolume: __4___ l CDW total:__53.2___ g REVISION ========= Version 1 generated on 15.10.2008 from Striedner