strain/background: C57BL/6J tissue: hippocampus treatment: hypothermia (HT) time point: 56 days
Treatment protocol
For eFS, hyperthermia (HT) in mice was induced by quickly raising core body temperature with a temperature-regulated laminar stream of warm air (41-48ºC) as previously described (Van Gassen/Hessel 2008 Genes Brain Behav.). HT was typically reached within 2.5 minutes. After core temperature of the HT mice had reached 42ºC, air temperature was adjusted to maintain a core body temperature between 41.5ºC and 42ºC. Time and temperature data was logged automatically on a computer (DASHost v1.0, Plexx). HT was maintained for 30 minutes. NT mice were simultaneously treated as HT mice, except that the core body temperature of the animals was kept at 35ºC. The observer scored the stages of behavior and seizures during the experiment, as previously described (van Gassen/Hessel 2008, Genes Brain Behav.). For eFS, only mice showing continued febrile seizures during at least 17.5 minutes were used in the experiment. At one hour, three, fourteen, and fifty-six days after HT, animals were sacrificed by decapitation. Brains were quickly removed and hippocampal tissue was dissected, collected and stored at -80ºC. Total RNA was isolated, purified and checked for quality as described (van Gassen 2007 Epilepsia).
Growth protocol
Mice were bred from pairs of C57BL/6J mice (Jackson Laboratories, ME, USA) and raised by their mothers. Animals were kept in a controlled 12h light-dark cycle with a temperature of 22 ± 1ºC and were given unrestricted access to food (211 RMH-TM diet; Hope Farms, Woerden, NL) and water. Animals were housed in transparent Plexiglas cages with wood-chip bedding and tissue for nest building.
Extracted molecule
total RNA
Extraction protocol
Trizol RNA isolation of tissue sections: 1. After sectioning, store sections in ice-cold RNAlater (~10 ml RNAlater per 20-mm section). Make sure that the sections do not thaw before they are in ice-cold RNAlater. 50 ml RNAlater. 2. Add X ml Trizol (10x volume of RNAlater) and mix by inverting the tube several times. 500 ml Trizol. 3. Incubate 5 min at room temperature. 4. Pre-incubate Proteinase K (10 mg/ml) at 37 C for at least 10 minutes. 5. Add 10 ml of the pre-heated Proteinase K solution to Trizol mixture. 10 ml Prot-K. 6. Incubate 30 min at 55 C. 7. Add X ml chloroform (20 ml per 100 ml Trizol), mix for 15 seconds by shaking vigorously. Incubate 2-3 min at room temperature. 100 ml chloroform. 8. Centrifuge 20 min at 14000 rpm without brake at 4 C. After centrifugation, transfer the water-phase to a new 1.5 ml micro centrifuge tube (~300 ml). 9. Continue with RNeasy columns (Qiagen) for clean-up of RNA. 10. Adjust each sample to a volume of 300 ml with sterile MilliQ water. Add 160 ml buffer RLT to the samples, and mix (= lysis buffer). 11. Add 240 ml ethanol (100% stored at -20 C) to the lysate, mix well by pipetting and immediately apply the sample to an RNeasy mini spin column sitting in a 2 ml collection tube (use a second P1000 pipette and work as quickly as possible). Next lysate, and so on. 12. Centrifuge at 8000 rpm for 15 seconds in an Eppendorf centrifuge. Discard flow-through and collection tube. 13. Transfer the RNeasy column into a new 2 ml collection tube. Add 500 ml buffer RPE, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse the collection tube in step 5. 14. Pipette 500 ml buffer RPE onto the RNeasy column, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse collection tube. 15. Centrifuge at 10,000 rpm for 2 minutes (to dry the RNeasy membrane). 16. Transfer the RNeasy column into a new 1.5 ml collection tube. Pipette 100 ml milliQ directly onto the RNeasy membrane and incubate for 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. 17. Pipette the eluate of step 7 again onto the RNeasy membrane and incubate for another 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute.
Label
Cy5
Label protocol
aRNA labeling with Cy-dyes 1· In dark micro centrifuge tube; dilute/concentrate required aRNA sample (x ng) to 8 µl. 2· Add 1 µl 0.5 M NaBicarbonate buffer, pH 9. Mix well. 3· Resuspend monofunctional NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): Quickly resuspend entire tube in 10 µl DMSO (Merck 8.02912.10). Add 1.25 µl per sample. Mix immediately and quickly. 4· Incubate 60 min at RT in the dark. 5· Add 4.5 µl 4 M hydroxylamine for quenching to prevent cross-coupling. 6· Incubate 15 min at RT in the dark. Cleanup using Clontech ChromaSpin-30 columns: 1. Upon removing a CHROMA SPIN Column from the protective plastic bag, invert it several times to resuspend the gel matrix completely. Before removing the top make sure that all the matrix is at the bottom by vigorously flicking the column downwards to remove matrix that is stuck to the top cap. 2. Holding the CHROMA SPIN Column upright, grasp the break-away end between your thumb and index finger and snap off. Place the end of the spin column into one of the 2-ml microcentrifuge (collection) tubes provided, and lift off the top cap. Save the top cap and the white-end cap. 3. Centrifuge at 700 g for 5 min (2700 RPM in Heraeus Megafuge 1.0 Sepatech Centrifuge, 1500 RPM in Eppendorf 5810 centrifuge (prog. 5)). After centrifugation, the column matrix will appear semi-dry. This step purges the equilibration buffer from the column and re-establishes the matrix bed. Make sure that the matrix bed is flat. 4. Remove the spin column and collection tube from the centrifuge rotor, and discard the collection tube and column equilibration buffer. 5. Place the spin column into the second 2-ml microcentrifuge tube. Carefully and slowly apply the sample to the center of the gel bed's flat surface. Drop by drop! Do not allow any sample to flow along the inner wall of the column - this will completely mess up the sample. 6. Centrifuge at 700 g for 5 min. 7. Remove the spin column and collection tube from the rotor and detach them from each other. The purified sample is at the bottom of the collection tube. Immediately keep on ice and in the dark! Purified sample is about 15-30 µl. Bring sample up to 35 µl with water and take 5 µl eluate for spectrophotometry (190 to 750 nm range). Samples may be stored at -20 C.
strain/background: C57BL/6J tissue: hippocampus treatment: normothermia (NT) time point: 56 days
Treatment protocol
For eFS, hyperthermia (HT) in mice was induced by quickly raising core body temperature with a temperature-regulated laminar stream of warm air (41-48ºC) as previously described (Van Gassen/Hessel 2008 Genes Brain Behav.). HT was typically reached within 2.5 minutes. After core temperature of the HT mice had reached 42ºC, air temperature was adjusted to maintain a core body temperature between 41.5ºC and 42ºC. Time and temperature data was logged automatically on a computer (DASHost v1.0, Plexx). HT was maintained for 30 minutes. NT mice were simultaneously treated as HT mice, except that the core body temperature of the animals was kept at 35ºC. The observer scored the stages of behavior and seizures during the experiment, as previously described (van Gassen/Hessel 2008, Genes Brain Behav.). For eFS, only mice showing continued febrile seizures during at least 17.5 minutes were used in the experiment. At one hour, three, fourteen, and fifty-six days after HT, animals were sacrificed by decapitation. Brains were quickly removed and hippocampal tissue was dissected, collected and stored at -80ºC. Total RNA was isolated, purified and checked for quality as described (van Gassen 2007 Epilepsia).
Growth protocol
Mice were bred from pairs of C57BL/6J mice (Jackson Laboratories, ME, USA) and raised by their mothers. Animals were kept in a controlled 12h light-dark cycle with a temperature of 22 ± 1ºC and were given unrestricted access to food (211 RMH-TM diet; Hope Farms, Woerden, NL) and water. Animals were housed in transparent Plexiglas cages with wood-chip bedding and tissue for nest building.
Extracted molecule
total RNA
Extraction protocol
Trizol RNA isolation of tissue sections: 1. After sectioning, store sections in ice-cold RNAlater (~10 ml RNAlater per 20-mm section). Make sure that the sections do not thaw before they are in ice-cold RNAlater. 50 ml RNAlater. 2. Add X ml Trizol (10x volume of RNAlater) and mix by inverting the tube several times. 500 ml Trizol. 3. Incubate 5 min at room temperature. 4. Pre-incubate Proteinase K (10 mg/ml) at 37 C for at least 10 minutes. 5. Add 10 ml of the pre-heated Proteinase K solution to Trizol mixture. 10 ml Prot-K. 6. Incubate 30 min at 55 C. 7. Add X ml chloroform (20 ml per 100 ml Trizol), mix for 15 seconds by shaking vigorously. Incubate 2-3 min at room temperature. 100 ml chloroform. 8. Centrifuge 20 min at 14000 rpm without brake at 4 C. After centrifugation, transfer the water-phase to a new 1.5 ml micro centrifuge tube (~300 ml). 9. Continue with RNeasy columns (Qiagen) for clean-up of RNA. 10. Adjust each sample to a volume of 300 ml with sterile MilliQ water. Add 160 ml buffer RLT to the samples, and mix (= lysis buffer). 11. Add 240 ml ethanol (100% stored at -20 C) to the lysate, mix well by pipetting and immediately apply the sample to an RNeasy mini spin column sitting in a 2 ml collection tube (use a second P1000 pipette and work as quickly as possible). Next lysate, and so on. 12. Centrifuge at 8000 rpm for 15 seconds in an Eppendorf centrifuge. Discard flow-through and collection tube. 13. Transfer the RNeasy column into a new 2 ml collection tube. Add 500 ml buffer RPE, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse the collection tube in step 5. 14. Pipette 500 ml buffer RPE onto the RNeasy column, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse collection tube. 15. Centrifuge at 10,000 rpm for 2 minutes (to dry the RNeasy membrane). 16. Transfer the RNeasy column into a new 1.5 ml collection tube. Pipette 100 ml milliQ directly onto the RNeasy membrane and incubate for 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. 17. Pipette the eluate of step 7 again onto the RNeasy membrane and incubate for another 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute.
Label
Cy3
Label protocol
aRNA labeling with Cy-dyes 1· In dark micro centrifuge tube; dilute/concentrate required aRNA sample (x ng) to 8 µl. 2· Add 1 µl 0.5 M NaBicarbonate buffer, pH 9. Mix well. 3· Resuspend monofunctional NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): Quickly resuspend entire tube in 10 µl DMSO (Merck 8.02912.10). Add 1.25 µl per sample. Mix immediately and quickly. 4· Incubate 60 min at RT in the dark. 5· Add 4.5 µl 4 M hydroxylamine for quenching to prevent cross-coupling. 6· Incubate 15 min at RT in the dark. Cleanup using Clontech ChromaSpin-30 columns: 1. Upon removing a CHROMA SPIN Column from the protective plastic bag, invert it several times to resuspend the gel matrix completely. Before removing the top make sure that all the matrix is at the bottom by vigorously flicking the column downwards to remove matrix that is stuck to the top cap. 2. Holding the CHROMA SPIN Column upright, grasp the break-away end between your thumb and index finger and snap off. Place the end of the spin column into one of the 2-ml microcentrifuge (collection) tubes provided, and lift off the top cap. Save the top cap and the white-end cap. 3. Centrifuge at 700 g for 5 min (2700 RPM in Heraeus Megafuge 1.0 Sepatech Centrifuge, 1500 RPM in Eppendorf 5810 centrifuge (prog. 5)). After centrifugation, the column matrix will appear semi-dry. This step purges the equilibration buffer from the column and re-establishes the matrix bed. Make sure that the matrix bed is flat. 4. Remove the spin column and collection tube from the centrifuge rotor, and discard the collection tube and column equilibration buffer. 5. Place the spin column into the second 2-ml microcentrifuge tube. Carefully and slowly apply the sample to the center of the gel bed's flat surface. Drop by drop! Do not allow any sample to flow along the inner wall of the column - this will completely mess up the sample. 6. Centrifuge at 700 g for 5 min. 7. Remove the spin column and collection tube from the rotor and detach them from each other. The purified sample is at the bottom of the collection tube. Immediately keep on ice and in the dark! Purified sample is about 15-30 µl. Bring sample up to 35 µl with water and take 5 µl eluate for spectrophotometry (190 to 750 nm range). Samples may be stored at -20 C.
Hybridization protocol
PREHYBRIDIZATION: Make 100 ml prehybridization buffer (for a maximum of 4 slides) containing 5xSSC, 25% formamide (Merck Cat# 1.09684.10), 0.1% SDS and 1% bovine serum albumin (BSA; Sigma Cat# A-9418): 25 ml 20x SSC, 25 ml pure formamide, 49 ml ddH2O, 1 ml 10% SDS and 1 g BSA. After BSA has dissolved the solution becomes clear in about 5 minutes. Filter prehybridization buffer through a 0.22 micron syringe filter. Heat the prehybridization solution to 42C in a 100 ml Coplin Jar. Place slides to be analyzed into the preheated prehybridization buffer. Incubate for 45 minutes at 42C. Wash the slides by dipping 5 times in room temperature ddH2O (staining dish). Dip the slides 5 times in room temperature isopropanol (staining dish). Air dry in fume hood (15 ml tube rack upside down). Slides should be used immediately following prehybridization. HYBRIDIZATION: (NOTE: The first steps of the hybridization protocol can be completed during the prehybridization step.) The hybridization volume for a whole slide (25x60mm) is 45 ul. Maximum volume of Cy3 and Cy5 combined target is 25 ul! It could be necessary to concentrate in a speed vac until the volume is below 25 ul. Prepare 2x hybridization buffer containing 50% formamide, 10xSSC and 0.2% SDS: 2.5 ml formamide, 2.5 ml 20xSSC and 0.1 ml 10% SDS. Immediately filter the hybridization buffer through a 0.22 micron filter (before SDS precipitates out of solution). Take 250 ul of the 2x hybridization buffer. Add 5 ul Herring sperm DNA (stock 10 ug/ul, sheared). Final concentration in 2x hybridization buffer is 200 ug/ml. In case of Human Gene Arrays add also 5 ul tRNA (stock 20 ug/ul). Preheat to 42C to overcome SDS precipitation. Combine 25 ul target with 25 ul 2x hybridization buffer (preheated to 42C). Heat at 95C for 5 minutes. Spin at 12K for 2 minutes. DO NOT place on ice after the 2 minute spin. Using standard coverslips: To each array add a total of 45 ul target as 3 to 6 separate dots in the center, along the length of the slide. Put a coverslip (25x60mm) on the array by holding the coverslip in place with your fingertips, toward the bottom of the slide. Gently (!) allow the coverslip to drop onto the array by using a needle. Do not move the coverslip after it is in place. Using LifterSlips (Erie Scientific): Place clean LifterSlip, teflon side down, over the array area. Place the pipette tip along an open edge of the LifterSlip and slowly pipette 45 ul target out of the tip. Capillary action will draw the probe mix under the slip. Place the slide in a Corning Hybridization Chamber (Corning Cat# 2551). Add 20 ul of water to each well in the chamber and clamp the hybridization chamber closed. Gently place the hybridization chamber into a 42C water bath for 16-20 hours. WASHING THE HYBRIDIZED ARRAYS: Remove the slide from the hybridization chamber, taking care not to disturb the coverslip. Perform the following washes: (i) Low-stringency wash: Place the slides in a Coplin Jar containing 100 ml 1xSSC and 0.2% SDS (= 100 ml 1xSSC and 2 ml of 10% SDS). Gently remove the coverslip while the slide is in solution. LifterSlips are reusable and should be washed in 70% EtOH prior to use. Incubate for 4 minutes at room temperature. (ii) High-stringency wash: Place the slides in a Coplin Jar containing 100 ml 0.1xSSC and 0.2% SDS (= 10 ml 1xSSC, 90 ml water and 2 ml of 10% SDS). Incubate for 4 minutes at room temperature. Wash the slide finally in 100 ml 0.1xSSC to remove particles of SDS. Incubate for 4 minutes at room temperature. Allow the slides to dry by centrifuge 1 minute 500 rpm (do this immediately after the third wash).
Scan protocol
Scanning of slides using the Agilent G2565BA scanner. Features were extracted using ImaGene software from BioDiscovery.
Description
HT-induced eFS.
Data processing
Normalization was done using print-tip LOESS as described in (Yang et al. 2002), for all gene probes using no background subtraction and a span of 0.4. Probes flagged as absent, or with a (nearly) saturated signal (i.e., > 2^15) in either channel were not considered for the estimation of the LOESS curve. Signals were corrected for dye bias using intrinsic gene-specific dye biases estimated from the hybset itself, as described in Margaritis, Lijnzaad et al., Mol. Sys. Biol. 2009.
