bone marrow macrophages from C57BL/6 mice aged 6-8 weeks
Biomaterial provider
Medzhitov Lab
Treatment protocol
Macrophages were left untreated (Naïve) or stimulated with 100ng/ml LPS for 24 hours (Tolerant), washed twice with warm PBS and given fresh media (N, T) or 10ng/ml LPS (N+L, T+L) for 4 hours.
Growth protocol
Bone marrow progenitors were harvested from mice and cultured for 7 days on petri dishes in M-CSF supplemented RPMI-1640. Cells were lifted with cold PBS and replated on tissue-culture treated plates.
Extracted molecule
total RNA
Extraction protocol
Total RNA from BMMΦ was isolated with RNA-bee reagent (Tel-Test) according to the manufacturer's instructions.
Label
biotin
Label protocol
Total RNA from each sample was used to prepare biotinylated target RNA. Briefly, 10 µg of total RNA was used to generate first-strand cDNA by using a T7-linked oligo(dT) primer. After second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in approximately 100-fold amplification of RNA.
Hybridization protocol
Target cDNA generated from each sample were then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System. Briefly, spike controls were added to 10 µg fragmented cRNA before overnight hybridisation. Arrays were then washed and stained with streptavidin-phycoerythrin.
Scan protocol
Arrays were scanned on an Affymetrix GeneChip scanner 3000.
Description
Additional protocols:
cDNA SYNTHESIS
FIRST STRAND cDNA SYNTHESIS
Important notes:
• Step 1. The recommended starting amount of total RNA is 10µg per reaction. Do not use less than this. 10µg of total RNA produces approximately 40µg to 110µg of cRNA. If you require more cRNA than this then 2 reactions for each sample should be carried out. • Step 3 breaks the secondary structure of RNA and step 4 prevents secondary structure reformation. These steps are the key steps for successful cDNA preparation. • The T7(dT)24 primer used in the cDNA synthesis must be of high quality and HPLC-purified. Insufficiently purified primer may appear to produce good double stranded cDNA, (since the 5’ end of the primer is not critical for the priming step), but still result in poor in vitro transcription yields if the primer is contaminated with shorter sequences, (missing the 5’ end). Affymetrix have found GENSET’s HPLC-purified T7(dT)24 primer to be very reliable. Affymetrix also sells the T7(dT)24 primer as a quality assured kit (T7-Oligo dT Promoter Primer Kit ) • A master mix should be prepared for step 5. Mix thoroughly, spin briefly if necesssary.
METHOD
1. Place the following in a microfuge tube:
X µl (10µg) total RNA in DEPC treated dH2O. 2 µl (50 µM) T7(dT)24 primer. Y µl DEPC treated dH2O. 11µl Total
2. Mix by pipetting, spin briefly if necessary. 3. Incubate at 65-70 0C for 10 minutes in a heated block 4. Place tubes on ice. 5. Prepare a master mix on ice. The quantities for each reaction are:
4µl (5x ) First Strand Buffer (thaw at 37 0C, keep on ice ) 2µl DTT (0.1M) 1µl dNTPs (10mM) 7µl Total
6. Mix by pipetting, spin briefly if necessary. Add 7µl of master mix to each reaction tube. 7. Incubate at 42 0C for 2 mins in a heated block.
8. Add 2µl Superscript II Reverse Transcriptase (200U/µl ). 9. Incubate at 42 0C for 1 hour in a heated block. 10. Place tubes on ice and proceed to ‘Second Strand Synthesis’ or freeze the samples on dry ice and store at -80 o C.
SECOND STRAND cDNA SYNTHESIS
Important notes:
• Exactly 2U of RNase H should be used per reaction. Check concentration. • Make a master mix for step 2. Mix thoroughly and spin briefly if necessary. • For step 5, incubate either in a water bath in the cold room or in a refrigerated block with a lid to reduce condensation. • For step 9, store at -80 0C only if you cannot proceed to ‘Clean-up of double stranded cDNA’ immediately after the synthesis is complete.
METHOD
1. Place all reagents and the first strand reaction tubes on ice. 2. Prepare a master mix on ice in a new microfuge tube. For each reaction use:
91µl DEPC treated dH2O 30µl Second Strand Buffer (5X ) 3µl dNTPs (10mM ) 1µl E. coli DNA Ligase 10U/µl (10U ) 4µl E. coli DNA Polymerase I 10U/µl (40U ) 1µl E. coli RNAse H 2U/µl (2U ) 130µl Total
3. Mix by vortexing, spin briefly if necessary. Add 130µl of master mix to each of the first strand reaction tubes. 4. Mix by pipetting, spin briefly if necessary. 5. Incubate at 16 0C for 2 hours. 6. Add 2µl (10U) T4 DNA Polymerase. 7. Incubate at 16 0C for 5 mins 8. Add 10µl 0.5M EDTA 9. Place tubes on ice and proceed to ‘Clean-up of double stranded cDNA’ or freeze the samples on dry ice and store at -80 0C.
CLEAN-UP OF DOUBLE STRANDED cDNA
Important points:
• The cDNA is cleaned using the GeneChip Sample Cleanup Module (Affymetrix). • Read the kit protocol before use and follow all the safety instructions. • The cDNA wash buffer is supplied as a concentrate. Before using for the first time, add 24ml of ethanol (96-100%) to obtain a working solution. • Perform all steps at room temperature and work without interruption. • Carry out centrifugation at >10,000 rpm. • After clean-up, all of the double stranded cDNA is used for the in vitro transcription reaction. • Do not attempt to quantify the cDNA as the primer can contribute significantly to the absorbance at 260nm.
METHOD
1. Add 600µl cDNA binding buffer to the 162µl cDNA preparation in a 1.5ml microfuge tube and mix by vortexing for 3 seconds. 2. Check that the colour of the mixture is yellow. If the colour is orange or violet, add 10µl of 3M sodium acetate pH 5.0 and mix to turn the solution yellow. 3. Apply 500µl of the sample to the cDNA Cleanup Spin Column sitting in a 2ml collection tube and spin for 1 min in a microfuge (>10,000rpm). Discard flow-through. 4. Reload the spin column with the remaining mixture (262µl) and spin. Discard flow-through and collection tube. 5.Transfer spin column to a new 2ml collection tube (supplied). 6. Pipette 750µl cDNA Wash Buffer onto the spin column and spin for 1 min in a microfuge (>10,000rpm). Discard flow-through. 7. Cut the cap off the spin column and spin for 5 mins at maximum speed to dry the membrane. Discard flow-through and collection tube. 8. Transfer spin column to a 1.5ml collection tube and pipette 14µl of cDNA Elution Buffer directly onto the spin column membrane. 9. Incubate for 4 mins at room temperature then spin for 1 min at maximum speed to elute. 10. Re-apply eluate to the spin column and repeat step 9. The average volume of eluate is 12µl from 14µl of elution buffer. 11. Place tubes on ice and proceed to the in vitro transcription reaction or freeze the samples on dry ice and store at -80 o C.
SYNTHESIS OF cRNA - IN VITRO TRANSCRIPTION.
Important points.
• The reagents used for the in vitro transcription are from the Enzo BioArray High Yield Transcript Labelling Kit. • Once thawed, reagents should be kept at room temperature until the reaction is ready to be incubated at 37 0C in order to reduce precipitation of DTT. • The reaction should be carried out in an incubator or warm room to reduce condensation on the inside of the lid.
METHOD
1. Thaw all reagents and double stranded cDNA at room temperature. 2. Prepare a master mix at room temperature. For each reaction use:
Add 28µl to each of the cDNAs previously prepared (usually 12µl).
3. Mix by pipetting and spin briefly if necessary. 4. Incubate at 37 0C for 5 hours. Gently mix the reaction every hour by tapping the tube. Spin briefly if necessary. 5. Proceed to ‘Clean-up of cRNA’ or freeze on dry ice and store at -80 0C.
CLEAN-UP OF cRNA
Important points:
• The cRNA is cleaned using the GeneChip Sample Cleanup Module (Affymetrix) to remove unincorporated dNTP which absorb at 260nm and would interfere with quantification of the cRNA. • IVT cRNA Wash Buffer is supplied as a concentrate. Before using for the first time, add 20ml of ethanol (96-100%) to obtain a working solution. • IVT cRNA Binding Buffer may form a precipitate on storage that can be redissolved by warming in a water bath at 300C then placing at room temperature. • Perform all steps at room temperature and work without interruption. • Do not touch the membrane of the spin column with the pipette tip. • Carry out centrifugation at >10,000 rpm.
METHOD
1. Add 60µl of RNase-free water to the in vitro transcription reaction and mix by vortexing for 3 secs. 2. Add 350µl of IVT cRNA Binding Buffer and mix by vortexing for 3 secs. 3. Add 250µl ethanol (96-100%) to the lysate and mix by pipetting. Do not spin. 4. Apply sample (700µl) to the IVT cRNA Cleanup Spin Column sitting in a 2ml collection tube. 5. Spin for 15 secs at >10,000rpm. Discard flow-through and collection tu
Data processing
Data was processed using Affymetrix Microarray Suite version 5.0, scaled to a target intensity of 500.
