Investigation Title Transcription profiling of H2O2 treated rat cardiac myocytes
Comment[Submitted Name] EXP_TKAC_0102_01
Experimental Design dose_response_design compound_treatment_design time_series_design transcription profiling by array
Experimental Design Term Source REF mo EFO
Comment[ArrayExpressReleaseDate]
Comment[AEMIAMESCORE] 5
Comment[ArrayExpressAccession] E-MIMR-12
Comment[MAGETAB TimeStamp_Version] 2011-08-22 18:29:33 Last Changed Rev: 14857
Experimental Factor Name MO:timepoint MO:Compound
Experimental Factor Type timepoint compound_treatment_design
Experimental Factor Term Source REF
Person Last Name unknown Clerk Kemp
Person First Name unknown Angela Tim
Person Mid Initials
Person Email derek.fowler@ic.ac.uk a.clerk[at]imperial.ac.uk t.kemp[at]imperial.ac.uk
Person Phone +44 (0)20 8383 2405 n/a n/a
Person Fax +44 (0)20 8383 8577
Person Address CSC/IC Microarray Centre Hammersmith Campus, L-block Du Cane Road, London UK
Person Affiliation Imperial College South Kensington Campus Imperial College South Kensington Campus
Person Roles submitter submitter
Person Roles Term Source REF
Quality Control Type
Quality Control Term Source REF
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date 2011-11-08
PubMed ID
Publication DOI
Publication Author List Institute of Laboratory Animal Resources, National Research Council
Publication Title Animals for Research: A Directory of Sources
Publication Status
Publication Status Term Source REF
Experiment Description Title: Changes in gene expression affected by H2O2 in cardiac myocytes.
Description: We aim to identify the changes in gene expression in response to
oxidative stress in rat neonatal ventricular myocytes.
Oxidative stress will be induced by dosing neonatal ventricular myocyte
cultures with 0.2, 0.1 and 0.04mM hydrogen peroxide at 2, 4 and 8 hr time
points using unstimulated myocytes as control.
Protocol Name csc.mrc.ac.uk:mimir/Protocol:RNAzol B csc.mrc.ac.uk:mimir/Protocol:myocyte_prep csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_01o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_01n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_01m csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_01q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_02m csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_02n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_02o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_02q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_10m csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_10o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_10q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_10n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_05m csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_05o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_05n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_05q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_03m csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_03n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_03o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_03q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_04m csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_04n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_04o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_04q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_08m csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_08n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_08o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_08q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_06q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_06m csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_06n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_06o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_07m csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_07o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_07q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_07n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_09q csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_09n csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_09o csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_09m csc.mrc.ac.uk:mimir/Protocol:cRNA_prep_phenol csc.mrc.ac.uk:mimir/Protocol:cRNA_frag csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_23p csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_31p csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_26p csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_24p csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_27p csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_28p csc.mrc.ac.uk:mimir/Protocol:BSM_TKAC_30p csc.mrc.ac.uk:mimir/Protocol:hyb_cocktail csc.mrc.ac.uk:mimir/Protocol:scan_1 Affymetrix:Protocol:ExpressionCall
Protocol Type nucleic_acid_extraction grow specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action labeling specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action hybridization image_acquisition bioassay_data_transformation
Protocol Description Title: Total RNA preparation (RNAzol B). Description: RNAzol" B
Isolation of RNA
1. Introduction
The RNAzol" and RNAzol" B methods, which have been formulated as a result of extensive laboratory investigation, are both based on the unique property of RNAzol" which promotes formation al complexes of RNA with guanidinium and water molecules, and abolishes hydrofilc interactions of DNA and proteins. In effect, DNA ard proteins are efficiently removed from the aqueous phase, while RNA remains in this phase during the sample extraction with RNAzol". The RNAzol" method can be completed within 3 hours, and the RNAzol" B method within 1.5 hour. Both methods provide the same high yield and purity of RNA preparations. The simplicity of the RNAzol" B mothods and excellent recovery of RNA from small quantities of tissue or cells makes our products especially suitable far gene expression studies for which only a limited quantity of biological material is available. The RNAzol" is undegraded, free of DNA and proteins and contains the whole spectrum of RNA molecules, including small (4-5 S) RNAs. The preparation is ready for dot blot hybridization, gel electrophoresis to detect specific mRNA by Northern blotting, poly A+ selection by the oligo dT-cellulose method, or may be used for molecular cloning, in vitro translation, PCR*, RNase protection and other enzymatic assays. The RNAzol" methods may also be used for rapid and efficient removal of DNA from RNA probes used in hybridization assays.
2. Reagent supplied
RNAzol" B: 1 bottle (100, 200 or 500 mL) containing a solution of RNAzol" B
Preparation: Ready to use.
Storage: Refrigerate at 2- 8 M-BM-0C. Do not freeze.
Stability: Refer to expiration date on the bottle (Stable up to nine months).
3. Reagent required, but not supplied
Chloroform (ACS grade)
lsopropanol (ACS grade)
75% Ethanol (ACS grade)
4. Method
The RNAzol" B method includes the following steps:
1. Homogenization RNAzol" B (2 mL/1O0 mg tissue or 10x10 cells)
2. RNA Extraction 1 vol. homogenate + 0.1 vol. Chloroform
3. RNA Precipitation 1 vol. isopropanol
4. RNA Wash 75% ethanol
Unless stated otherwise the procedure is carried out at room temperature.
4.1 Homogenization
Homogenize tissue samples with RNAzol" B (2 mL per 100 mg tissue> with a few strokes in a glass-Teflon homogenizer.
To isolate RNA from cells grown in suspension, sediment cells and lyse them by the addition of 0.2 mL of RNAzol" B per 106 cells. Cells grown in manolayer are lysed directly in the culture dish by the addition al RNAzol" B (1 mL per 3.5 cm petri dish or 10 cm2 (ex. T-30 flask = 30 cm2 = 3 mL). Solubilize RNA by passing the lysate a few times through the pipette.
4.2 RNA extraction
Add 0.2 mL chloroform per 2 mL of homogenate, cover the samples tightly, shake vigorously for 15 seconds (do not vortex) and let them stay on ice (or at 4 M-BM-0C for 5 minutes. Centrifuge the suspension at 12,00Og (4 M-BM-0C) for 15 minutes.
After addition of chloroform and centrifugation, the homogenate forms two phases: the lower blue phenol-chloroform phase and the colorless upper aqueous phase whereas DNA and proteins are in the interphase and arganic phase. A volume af the aqueous phase is about 50% of the initial volume of RNAzol" B plus a volume of tissue used for homogenization.
4.3 RNA precipitation
Transfer the aqueous phase to the fresh tube, add an equal volume of isapropanol and store the samples for 15 minutes at 4 M-BM-0C. Centrifuge samples for 15 minutes at 12,000 g (4 M-BM-0C). RNA precipitate (often invisible before centrifugation) forms a white-yellow pellet at the bottom al the tube.
4.4 RNA wash
Remove the supernatant and wash the RNA pellet once with 75% ethanol by vortexing and subsequent centrifugation for 8 minutes at 7,500 g (4 M-BM-0C or 20 M-BM-0C). Use at least 0.8 mL of ethanol per 50-100 mg RNA. At the end of the procedure, dry the pellet briefly under vacuum far 10-15 minutes. It is important not to let the RNA pellet dry completely, as it will greatly decrease its solubility. Dissolve the RNA pellet in 0.5% SDS or in lmM EDTA, pH 7 solution by vortexing or by passing the RNA solution a few times through a pipette tip. An incubation far 10-15 minutes at 60 M-BM-0C may be required to dissolve preparations of RNA. Diethylpyrocarbonate (DEPC) - treated RNase free solutions (3) should be used for RNA solubilization. The final preparation is free of DNA and proteins and has a 260/280 ratio higher than 1.9.
5. Notes and comments
Isolation of RNA from a small amount of tissue (1-10 mg): Homogenize samples in 0.8 mL of RNAzol" B, transfer the homogenate to Eppendorf type tubes, add 80 ul of chloroform and store samples for 5 minutes at 4 M-BM-0C. Centrifuge samples in an Eppendorf centrifuge for 15 minutes, collect the aqueous phase and precipitate RNA with 0.4 mL of isopropanol for 45 minutes or overnight at 4 M-BM-0C. Centrifuge RNA precipitates for 15 minutes and wash once with 0.8 mL of 75% ethanol.
Isolation of RNA from blood, serum and other liquid matrices: Homogenize samples in 10- 15 mL of RNAzol" B per 1 mL of sample.
Isolation of RNA from bacterial cells. homogenize samples in 10-15 mL of RNAzol" B per 1 mL pellet of bacterial cells.
Following isopropanol addition, store samples overnight at 4 M-BM-0C in case the procedure has to be interrupted at this step.
An additional precipitation is necessary to use RNA isolated by the RNAzol" B method in enzymatic assays. Following solubilization, precipitate RNA in the presence of 0.2 M NaCI with one volume of isopropanol or with two volumes of ethanol for 1 hour at 20 M-BM-0C.
Hands and dust may be the major source of RNase contamination. Use gloves and keep tubes closed. The use of sterile, disposable polypropylene tubes is recommended throughout the procedure.
Some commercial SDS preparations have acid pH. Adjust SDS solution to pH 6.5 - 7.5 if necessary.
Title: Myocytes preparation from rat heart. Description: Neonatal Myocyte Prep Protocol
NB: 50 rats or more = 2 preps.
Following protocol is for one prep.
Checklist:
M-BM-7 Turn on shaking water bath and the small static waterbath.
M-BM-7 Warm DMEM, M199, penicillin and streptomycin mix, feotal and horse serums and 1% gelatin solution to 37M-BM-0C.
M-BM-7 Spray hood with 70% IMS and wipe.
M-BM-7 Make sure to have at hand a good quantity of sterile pipettes; 25ml, 10ml and 5ml pipettes; sterile Pasteur pipettes; tips; gloves; 50ml falcon tubes and 70% IMS.
M-BM-7 2 falcon tube racks.
M-BM-7 Waste pot with a small amount of bleach in.
M-BM-7 2 x 100ml autoclaved duran bottles. (1 for the hearts and 1 for the enzyme.)
M-BM-7 A sterilized glass petri dish with lid (baked, 2 h, 2250C).
M-BM-7 Sterilized dissection instruments (baked as above) in a large petri dish covered with foil.
M-BM-7 1 large ice bucket (with ice) for the dissection and 1 smaller one for the enzymes.
M-BM-7 Place the glass petri dish with lid (one for each prep) in the larger icebox. Place the lower part of a 60mm primaria dish upside down in the petri dish as a platform for the dissection. Add 15ml of 1M-BM-4ADS buffer to the petri dish.
M-BM-7 Place a 60mm primaria dish (one for each glass petri dish) on the ice too.
M-BM-7 Weigh out 24mg of collagenase (kept in the fridge) and 30mg of pancreatin (kept in the -200C freezer) into separate scintillation vials ^S place in the hood. NB: Double the amount of enzyme is used for two preps, but make sure to weigh out in another two vials as a vial can only hold 20ml.
M-BM-7 Add 20ml of 1M-BM-4ADS buffer to each vial. Vortex the vial containing pancreatin to dissolve powder completely.
M-BM-7 Filter the enzymes. Use the sterile bottle top filter and attach it to the pump via a piece of tubing. Affix the filter to a 100ml duran bottle, turn the pump on and add the enzyme solutions. Add another 5ml of 1M-BM-4ADS to wash out each vial and filter.
M-BM-7 Therefore, for one prep there will be a total of 50ml of enzyme solution.
Gelatin coating the dishes:
M-BM-7 Add approx 2ml of gelatin to each 60mm dish and 1ml to each 35mm dish, swirl to ensure the bottom of the plates are covered.
M-BM-7 Once all the dishes are coated, remove the gelatin and discard. The gelatin will take at least 2 h to dry.
Dissection:
M-BM-7 Have at hand: paper towels, 70% IMS and 2 foil covered polystyrene slabs (paper towels saturated with IMS placed on top.)
M-BM-7 Rats are killed and heads removed, sprayed with IMS then placed on the slabs.
M-BM-7 To dissect and remove the heart: Using the larger instruments, hold the front legs back with the curved forceps and use the scissors to make an incision from the neck through the sternum making sure the rib cage has been dissected.
M-BM-7 Hold the forceps closer together and press downwards on either side of the incision, the heart will pop out. Cut the heart out.
M-BM-7 Place the heart in the petri dish containing 1M-BM-4ADS buffer. Repeat with the other rats.
M-BM-7 NB: If there are more than 50 rats i.e. two preps, then make sure the two petri dishes contain the same number of hearts.
M-BM-7 After the dissection, place 5-10 hearts on the platform in the petri dish. Use the smaller instruments and cut away the feathery looking atria. Roughly chop the hearts into small pieces and transfer the pieces to the primaria dish. Repeat until all the hearts have been homogenized.
M-BM-7 Cut off the end of a Pasteur pipette, draw up the heart pieces and 1M-BM-4ADS buffer and transfer into a sterile 100ml duran bottle. Draw off the 1M-BM-4ADS buffer with a sterile Pasteur pipette by carefully tipping the duran and rotating it to separate the heart pieces from the buffer.
Digestion:
Digest Volume of enzyme soln/ml Time/mins Speed
1 10 5 9
2 10 20 7.5
3 8 25 6.5
4 8 25 6.5
5 6 15 7.5
6 6 10 6.5
M-BM-7 For the first digestion add 10mls of enzyme to the flask.
M-BM-7 Place into the shaking water bath at speed 9 for 5 mins.
M-BM-7 Remove from water bath and place in hood (spraying with IMS first.)
M-BM-7 Remove the liquid and discard into the waste pot.
M-BM-7 For the second digestion add 10mls of enzyme and follow the above steps but the water bath speed = 7.5 and the digestion time = 20 mins.
M-BM-7 Add the liquid, which is removed after the digestion, to 2mls of feotal calf serum (2 tubes are used if there are 2 preps) in a sterile 50ml falcon tube. The serum quenches the reaction of the enzymes.
M-BM-7 Centrifuge for 5 mins at 600rpm.
M-BM-7 Continue the digestions as above.
M-BM-7 Remove the tube from the centrifuge, carefully pipette off the supernatant and discard.
M-BM-7 Tap the pellet to loosen it and add 4mls of feotal calf serum, transfer this to another 50ml falcon tube and place in a rack in the incubator. Loosen the lid of the falcon tube to allow CO2 to reach the cell suspension. Repeat this for each digestion.
M-BM-7 After all the digestions, the cells are therefore collected in one falcon tube. Pool the cells from 2 preps if necessary into one falcon tube. Centrifuge at 600 rpm for 5 min.
M-BM-7 Carefully pipette off the supernatant, tap the pellet and resuspend in plating media. The amount of plating media used depends on the number of hearts in the prep (4ml for every 10 hearts).
M-BM-7
Pre-Plating (to remove adherent non-myocytes):
M-BM-7 Use 1 primaria dish per 10 hearts.
M-BM-7 Add 4ml of the suspension to each dish and place on a tray in the incubator for 30-40 mins.
Washing the pre-plates:
M-BM-7 The preplates are washed with plating media.
M-BM-7 E.g. If there are 4 preplate dishes labeled; 1, 2, 3 and 4. Remove the solution (this solution contains the myocytes that have been extracted) from plate 1 with a Pasteur pipette and transfer it to a 50ml falcon tube. Whilst removing it, pipette up and down a few times to wash the surface of the dish. Then add 4ml of plating media to dish 1.
M-BM-7 Remove the solution from dish 2 in the same way.
M-BM-7 Transfer the plating media from plate 1 to plate 2. Add 4ml of fresh plating media to dish 1. The bottle of plating media can now be placed back into the water bath.
M-BM-7 Remove the solution from dish 3 as above.
M-BM-7 Transfer the solution from dish 2 to dish 3 making sure dish 2 has been washed as above.
M-BM-7 Transfer the solution from dish 1 to dish 2 in the same way. Dish 1 can now been discarded.
M-BM-7 Continue until all the dishes have been washed in this way. The falcon tub now contains myocyte that are to be counted and plated out.
M-BM-7 NB: If fibroblasts are required, the preplate dishes should not be discarded, but another 4ml of plating media added to them and placed in the incubator.
Counting the cells:
M-BM-7 To a 1.5ml eppendorf tube; add 75ml of the cell solution in the falcon tube, 175ml of plating media and 250ml of Trypan Blue solution.
M-BM-7 Shake well then add to a haemocytometer using a gilson pipette, place under a microscope. Count the cells in the areas marked with an ^XX^Y. Only count the ^Xbeige^Y cells, blue ones are dead and should not be included. Total number of cells counted in the 5 areas = n.
M-BM-7
M-BM-7 Use the following equation to calculate the total number of myocyte cells extracted from the hearts.
M-BM-7
n x 4/3 x 103 x volume of cell solution in the falcon tube = total number of cells
M-BM-7 It can therefore be possible to calculate the number of cells extracted per heart and to calculate how many dishes are to be plated out, the number of cells for a 60mm dish is 4 million and for a 35mm dish it is 1.5 million.
Plating out the cells:
M-BM-7 The total amount of solution per 60mm dish to be plated out is 4ml and per 35mm dish is 2ml. The solution is made up of the cell suspension and fresh plating media. Therefore the volume of plating media must be calculated.
M-BM-7 Once made up the solution is added to the gelatin coated dishes. Swirl the plates to ensure the myocytes are evenly distributed over the plates.
M-BM-7 Plates are then put on stainless steel trays and placed in the incubator overnight.
Replacing the plating media with serum free maintenance media:
M-BM-7 Serum free maintenance media is warmed to 37M-BM-0C.
M-BM-7 The plating media is drawn off and discarded and is replaced with 4ml maintenance media per 60mm dish, 2ml per 35mm dish. The cells are then placed back in the incubator.
RECIPES
1M-BM-4ADS buffer
g per 1l
NaCl 6.8
HEPES 4.76
NaH2PO4 0.12
Glucose 1.0
KCl 0.4
MgSO4 0.1
Make up with MilliQ water and pH to 7.35 with NaOH, filter and store at 4M-BM-0C
1% Gelatin
5g in 500ml milliQ water, autoclave and store at 4M-BM-0C
Plating media
ml per 500ml
DMEM (#42430025, invitrogen) 340
M199 (#22340020, invitrogen) 85
Horse serum 50
Feotal calf serum 25
penicillin and streptomycin mix 5
Store at 4M-BM-0C
Maintenance media
ml per 500ml
DMEM (#42430025, invitrogen) 400
M199 (#22340020, invitrogen) 100
penicillin and streptomycin mix 5
Store at 4M-BM-0C
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0mM H2O2 for time0
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0mM H2O2 for time0
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0mM H2O2 for time0
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0mM H2O2 for time0
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0mM H2O2 for time0
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0mM H2O2 for time0
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0mM H2O2 for time0
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0mM H2O2 for time0
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.2mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.2mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.2mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.2mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time8
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time8
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time8
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time8
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time8
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time8
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time8
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time8
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.2mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.2mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.2mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.2mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.04mM H2O2 for time4
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
STEP 1: culture in medium with serum: Cells are cultured overnight in DMEM/medium 199(4:1,v/v), 10% (v/v) horse serum, 5% FCS, 100units/ml pen/strep at 37C, 5% CO2.
STEP 2: culture in serum free medium: Cells are cultured for 24hrs in serum free medium (DMEM/medium 199 (4:1,v/v), 100units/ml pen/strep) at 37C, 5% CO2.
STEP 3: treatment with H2O2: treatment of cells with 0.1mM H2O2 for time2
STEP 4: harvesting of cells: cells harvested
STEP 5: extraction of total RNA: extraction with RNAzol B
Title: cRNA preparation using phenol. Description: cRNA preparation using phenol
These protocols are based on those provided by Affymetrix and include modifications from the Whitehead Institute for Biomedical Research, MIT, Boston and the CSC/IC Microarray Centre.
A. First strand cDNA synthesis
Things to note:
1. It is very important to start with good quality, clean, total RNA. Acceptable ratios for the absorbance of the total RNA at 260nm and 280nm are between 1.9 and 2.1. Ensure that the total RNA is diluted, so that the absorbances measured are in the linear range for the spectrophotometer. This is often between 0.1 and 0.4 absorbance units.
2. For step (1) we recommend starting with 10mg total RNA per reaction tube. Do not start with less than 10M-BM-5g total RNA per reaction. Carry out two reactions for each sample if you require more than 40mg of cRNA.
3. Make a "master mix" where possible. Mix thoroughly and spin briefly, as necessary.
4. Use RNase free microfuge tubes and pipette tips throughout.
Method
1. Place the following in a microfuge tube:
- ml (10mg) total RNA in nuclease free treated dH2O
- ml (100 pmol) T7-(T)24 primer
(GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24)
1ml Poly A+ Controls
- ml nuclease free dH2O
11 ml final volume
2. Mix by pipetting, spin briefly as necessary.
3. Incubate at 65-70oC for 10 minutes.
4. Place tubes on ice.
5. Prepare a master mix on ice. The quantities are for each reaction/tube:
4ml (5x) First Strand Buffer (thaw at 37oC, put on ice), Invitrogen Y02321
2ml (0.1 M) DTT, Invitrogen Y00147
1ml (10mM) dNTPs, Invitrogen 18427-013
7M-BM-5l
Mix by pipetting, spin briefly as necessary. Add 7M-BM-5l of master mix to each reaction tube.
6. Incubate at 42 oC for 2 minutes.
7. Add 2ml Superscript II reverse transcriptase (400 U total), Invitrogen 18064- 014.
8. Tap tube to mix, spin briefly as necessary.
9. Incubate at 42oC for 1 hour in a waterbath.
10. Place tube on ice and proceed to "Second strand cDNA synthesis", or transfer tube to dry ice, allow reaction to freeze and store at -80oC.
B. Second strand cDNA synthesis
Things to note:
1. Check the units/M-BM-5l for the RNase H, it is important that exactly 2U are used per reaction; you may have to adjust volumes accordingly.
3. For step (4), incubate either in a water bath in the cold room or in a refrigerated block with a lid, to reduce condensation.
4. For step (5), store at -80oC only if you cannot go to the "Clean-up of double stranded cDNA" step immediately after the second strand cDNA synthesis is completed.
Method
1. Place all reagents and first strand reaction tubes on ice. Assemble master mix on ice.
2. Prepare a master mix in a new microfuge tube. For each reaction use:
- ?l nuclease free treated dH2O
30?l (5x) Second Strand Buffer, Invitrogen 10812-014
3?l (10mM) dNTPs, Invitrogen 18427-013
1M-BM-5l DNA Ligase (10 Units), Invitrogen 18052-019
4?l DNA Pol I (40 Units), Invitrogen 18010-025
- ?l RNase H (2 Units), Invitrogen 18021-071
130M-BM-5l
Mix by vortexing, spin briefly as necessary. Add 130?l of the master mix to each of the first strand reaction tubes.
3 Mix by pipetting, spin briefly as necessary.
4. Incubate at 16oC for 2 hours. Use a water bath in a cold room or a refrigerated block with lid, if possible. This reduces condensation.
5. Place tube on ice and proceed to "Clean-up of double stranded cDNA", or transfer tube to dry ice, freeze and store at -80oC.
C. Clean-up of double stranded cDNA
Things to note:
1. This method uses Phase-Lock tubes, which contain a gel that acts to separate the aqueous and organic layers in the phenol extraction permitting more complete recovery of the aqueous phase.
2. For step (1), spinning the Phase-Lock tubes allows the gel to settle to the bottom of the tube.
3. After the clean-up, ALL of the double stranded cDNA is used for the in vitro transcription reaction. Do not attempt to quantify the products of the reaction.
Method
1. Spin Phase-Lock tube in a microfuge at maximum (?12,000g) for 30 seconds.
2. To the cDNA reaction, add an equal volume (approx. 150 M-BM-5l) of buffer saturated phenol at room temperature (e.g. phenol-chloroform-isoamyl alcohol (25:24:1) Sigma P-2069 pH8.0).
3. Vortex briefly to mix.
4. Place the mixture into a Phase-Lock tube.
5. Spin at maximum (?12,000 g) in a microfuge for 2 minutes.
6. Transfer upper (aqueous) phase to a new tube
7. Add 0.5 x volume (7.5 M) NH4OAc (75 M-BM-5l), Sigma A-2706
4 M-BM-5l Glycogen (5 mg/ml), Ambion 9510
2.5 x volumes (100%) Ethanol (375 M-BM-5l)
8. Mix by tapping tube.
9. Spin at maximum at room temperature in a microfuge for 20 minutes - do not spin for longer.
10. Decant supernatant, being careful not to displace the pellet.
11. Add 160?l (80%) cold ethanol, spin at maximum for 5 minutes. Remove supernatant.
12. Add 160?l (80%) cold ethanol, spin at maximum for 5 minutes. Remove all the ethanol with a pipette (spin again briefly to collect any residual ethanol on the sides of the tube and remove ethanol a second time using a pipette).
13. Allow pellet to air dry for 5-10 minutes. Resuspend in 12?l of nuclease free dH2O.
14. Place tube on dry ice to freeze and then store at-80oC or proceed directly to the in vitro transcription reaction.
D. Preparation of cRNA
Things to note:
1. Once reagents have thawed, keep them at room temperature until incubation at 37oC to reduce precipitation of DTT.
2. The incubation should be carried out in an incubator or warm-room, to reduce condensation on the inside of the lid.
3. The reagents used for in vitro transcription are from the Bioarray High Yield RNA Transcript Labelling Kit, Enzo 900182.
Method
1. Thaw all reagents and double stranded cDNA at room temperature.
2. Prepare a master mix of the IVT reagents at room temperature. The quantities are for each reaction/tube:
12?l double stranded cDNA from previous method.
10?l nuclease free dH20
4?l (10x) HY Reaction Buffer
4?l Biotin-labelled ribonucleotides
4?l DTT
4?l RNase Inhibitor Mix
2?l T7 RNA Polymerase
40M-BM-5l
3. Mix by pipetting, spin briefly as necessary.
4. Incubate at 37oC for 5 hours. Gently mix the reaction every hour by tapping tube, spin briefly as necessary.
5. Proceed to "Clean-up of cRNA", or freeze by placing tube on dry ice and store at -80oC.
E. Clean-up of cRNA
Things to note:
1. Removal of unincorporated biotinylated ribonucleotides is very important obtaining a good quality scan.
2. cRNA clean up is carried out using the Qiagen RNeasy Mini Kit. Prepare reagents following the instructions in the kit. Add beta-mercaptoethanol to an aliquot of the RLT buffer shortly before use.
3. Be careful not to touch the tip of the spin column at any time.
4. Carry out centrifugation at M-BM-3 8,000g (M-BM-3 10,000rpm).
Method
1. Add to the in vitro transcription reaction tubes:
60ml nuclease free dH2O
350ml RLT buffer
2. Mix thoroughly by pipetting.
3. Add 250ml (100%) ethanol. Mix by pipetting, do not spin.
4. Apply sample to an RNeasy spin column.
5. Spin at maximum speed for 15 seconds.
6. Empty the collection tube.
7. Add 500ml RPE buffer.
8. Spin at maximum speed for 15 seconds.
9. Empty the collection tube.
10. Add 500ml RPE buffer.
11. Spin at maximum speed for 2 minutes.
12. Transfer spin column to a fresh tube.
13. Spin at maximum for 1 minute.
14. Transfer spin column to new collection tube, to prevent any carry-over of the RPE buffer.
15. Elute cRNA by placing 50ml nuclease free dH2O on the middle of the membrane of the spin column.
16. Incubate at room temperature for 4 minutes.
17. Spin at maximum for 1 minute.
18. Take the eluate and place on the membrane of the same spin column.
19. Incubate at room temperature for 4 minutes.
20. Spin at maximum for 1 minute.
21. Dilute an aliquot of the eluate with dH2O (not DEPC treated) and measure the absorbance at 260nm and 280nm. The dilution should be sufficient so that readings obtained are in the linear range for the spectrophotometer. Unless known otherwise assume this is between 0.1 and 0.4 absorbance units (try diluting 2ml of eluate in 198ml of dH2O). The cRNA should be clean, a ratio of the absorbances at 260nm to 280nm should be around 2.0, with an acceptable range of 1.9 to 2.1. You should have 40 to 110M-BM-5g of cRNA (assuming that you started with 10M-BM-5g total RNA where 1 OD260 unit is equivalent to 40M-BM-5g/ml single stranded RNA). If you have a yield of less than 40M-BM-5g of cRNA per reaction (per 10M-BM-5g reaction of total RNA), we recommend that you do not use the cRNA, as there is likely to be a problem with sample preparation.
22. Run a sample of the total RNA and cRNA for each sample (500ng to 1M-BM-5g) on a denaturing 1% agarose gel along with a marker spanning 200bp to 3kb (a DNA marker gives a rough idea of the size). The cRNA product should be a smear from about 100bp to 2kb with a brighter region from 500bp to 1kb, there is an example image at bottom of this protocol Photograph the gel, so that the markers are easy to identify.
23. Freeze by placing tube on dry ice and store at ^S80 M-BM-0C.
24. Bring the cRNA to the Microarray Centre on dry ice (after prior consultation with Peter Broderick or Helen Banks regarding a suitable time). Note that each sample should be clearly labelled on both the top and side of each tube with the date (day, month, year e.g. 05.11.01) and the sample identifier.
In order to assess the quality of the cRNA samples, prior to hybridisation on test arrays, we also require the following information, for both the cRNA samples and the total RNA from which it was generated.
(a) the raw absorbance readings
(b) the ratio of the absorbances at 260nm and 280nm
(c) the cRNA yield from 10M-BM-5g of total RNA
(d) an original photograph of the gel, with the markers and samples labelled.
Title: cRNA fragmentation. Description: Fragmentation of cRNA
Things to note:
1. This method provides enough fragmented cRNA to hybridise to one GeneChip. For rat and mouse samples three arrays, for human samples two U133 or five U95 arrays are required to cover the full set of genes and ESTs. Perform these reactions in parallel and combine before freezing.
2. Before addition, pulse-vortex for 30 seconds all reagents except for cRNA.
Method
1. Take RNase free microfuge tubes, one for each reaction.
2. To each tube add (at room temperature):
- ul cRNA (15ug)
13ul 5X fragmentation buffer
- ul RNase free dH2O
65ul final volume
3. Mix by pipetting, spin briefly as necessary.
4. Incubate at 95oC for 35 minutes.
5. Spin the fragmented cRNA reactions briefly to remove condensation from lid.
6. Combine the reactions for each sample (i.e. 3 for mouse/rat or 2/5 for humans).
7. Proceed directly to the hybridisation cocktail preparation.
STEP 1: pooled total RNA: pooled from BSM_TKAC_02m, n, o and q.
Note: BSM_TKAC_02m, n, o and q come from BS_TKAC_13, BS_TKAC_14, BS_TKAC_15 and BS_TKAC_16
respectively
STEP 2: cRNA preparation:
STEP 3: cRNA fragmentation: Fragmentation buffer lot # 2001/48
STEP 4: hyb cocktail preparation: 2X hyb buffer # 2001/64, Oligo B2 #1006611 , 20X Euk hyb # 1006550, Herring sperm DNA
# 13442202, Acetylated BSA #1109252
STEP 1: pooled total RNA: pooled from BSM_TKAC_10m, n, o and q.
Note: BSM_TKAC_10m, n, o and q come from BS_TKAC_13, BS_TKAC_14, BS_TKAC_15 and BS_TKAC_16
respectively
STEP 2: cRNA preparation:
STEP 3: cRNA fragmentation: Fragmentation buffer lot # 2001/48
STEP 4: hyb cocktail preparation: 2X hyb buffer # 2001/64, Oligo B2 #1006611 , 20X Euk hyb # 1006550, Herring sperm DNA
# 13442202, Acetylated BSA #1109252
STEP 1: pooled total RNA: pooled from BSM_TKAC_05m, n, o and q.
Note: BSM_TKAC_05m, n, o and q come from BS_TKAC_13, BS_TKAC_14, BS_TKAC_15 and BS_TKAC_16
respectively
STEP 2: cRNA preparation:
STEP 3: cRNA fragmentation: Fragmentation buffer lot # 2001/48
STEP 4: hyb cocktail preparation: 2X hyb buffer # 2001/64, Oligo B2 #1006611 , 20X Euk hyb # 1006550, Herring sperm DNA
# 13442202, Acetylated BSA #1109252
STEP 1: pooled total RNA: pooled from BSM_TKAC_03m, n, o and q.
Note: BSM_TKAC_03m, n, o and q come from BS_TKAC_13, BS_TKAC_14, BS_TKAC_15 and BS_TKAC_16
respectively
STEP 2: cRNA preparation:
STEP 3: cRNA fragmentation: Fragmentation buffer lot # 2001/48
STEP 4: hyb cocktail preparation: 2X hyb buffer # 2001/64, Oligo B2 #1006611 , 20X Euk hyb # 1006550, Herring sperm DNA
# 13442202, Acetylated BSA #1109252
STEP 1: pooled total RNA: pooled from BSM_TKAC_06m, n, o and q.
Note: BSM_TKAC_06m, n, o and q come from BS_TKAC_13, BS_TKAC_14, BS_TKAC_15 and BS_TKAC_16
respectively
STEP 2: cRNA preparation:
STEP 3: cRNA fragmentation: Fragmentation buffer lot # 2001/48
STEP 4: hyb cocktail preparation: 2X hyb buffer # 2001/64, Oligo B2 #1006611 , 20X Euk hyb # 1006550, Herring sperm DNA
# 13442202, Acetylated BSA #1109252
STEP 1: pooled total RNA: pooled from BSM_TKAC_07m, n, o and q.
Note: BSM_TKAC_07m, n, o and q come from BS_TKAC_13, BS_TKAC_14, BS_TKAC_15 and BS_TKAC_16
respectively
STEP 2: cRNA preparation:
STEP 3: cRNA fragmentation: Fragmentation buffer lot # 2001/48
STEP 4: hyb cocktail preparation: 2X hyb buffer # 2001/64, Oligo B2 #1006611 , 20X Euk hyb # 1006550, Herring sperm DNA
# 13442202, Acetylated BSA #1109252
STEP 1: pooled total RNA: pooled from BSM_TKAC_09m, n, o and q.
Note: BSM_TKAC_09m, n, o and q come from BS_TKAC_13, BS_TKAC_14, BS_TKAC_15 and BS_TKAC_16
respectively
STEP 2: cRNA preparation:
STEP 3: cRNA fragmentation: Fragmentation buffer lot # 2001/48
STEP 4: hyb cocktail preparation: 2X hyb buffer # 2001/64, Oligo B2 #1006611 , 20X Euk hyb # 1006550, Herring sperm DNA
# 13442202, Acetylated BSA #1109252
Title: Hybridisation cocktail preparation. Description: Hybridisation cocktail preparation
Things to note
1. Before addition, pulse vortex for 30 seconds all reagents.
Method
1. Heat 20x eukakaryotic hybridisation controls and control oligonucleotide B2 at 65oC for 5 minutes. Mix lightly by vortexing, spin briefly if necessary.
2. Prepare master mix, for each cRNA fragmentation reaction add:
150ul 2X hybridisation buffer
5ul control oligonucleotide B2 (3nM), Affymetrix 900301.
15ul 20X eukaryotic hybridisation controls
(BioB, BioC, BioD, cre) Affymetrix 900299.
3ul herring sperm DNA (10mg/ml), Promega D1811
3ul acetylated BSA (50mg/ml), Invitrogen 15561-020.
__ul RNase free dH2O
15ug cRNA
300ul Total Volume
3. Add an appropriate volume to the fragmentation reactions. Mix thoroughly by pulse-vortexing for 30 seconds.
4. Aliquot 300?l of fragmented cRNA into labelled tubes.
5. Proceed to ^\Hybridisation of fragmented cRNA^] or place fragmented cRNA on dry ice to freeze and store at ^S80oC.
Title: scanning on the old scanner settings. Description: 1. Switch on scanner.
2. Login to computer.
2. Launch Microarray Suite from desktop icon.
3. From ^\Tools^] menu select ^\Defaults^] and then ^\File Locations^] to check the location in which the files will be saved.
5 Open ^\Expts^] dialogue box. Complete as much information as possible and then save experiment. If performing wash and stain with antibody amplification, create 2 copies of each experiment. For one, mark the end of the experiment name with ba (before antibody); mark the other with aa (after antibody). Once all experiments have been entered close dialogue box.
4. Open ^\Scanner^] dialogue box, select experiment (ba), and then ^\Start^]. When instructed, load array into scanner. When scan is complete remove array from scanner.
5. Open ^\scanner^] dialogue box, select experiment (aa), and then ^\start^]. When instructed, load array into scanner. When scan is complete remove array from scanner.
6. Once all GeneChips are scanned proceed to ^XShutdown of GeneChip station^Y.
Title: ExpressionCall. Description: ExpressionCall
Protocol Parameters
Protocol Hardware
Protocol Software
Protocol Contact
Protocol Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology
SDRF File E-MIMR-12.sdrf.txt
Term Source Name mo The MGED Ontology nci_thesaurus ArrayExpress mo EFO The MGED Ontology
Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://nciterms.nci.nih.gov/NCIBrowser/Dictionary.do http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php
Term Source Version