|
| Status |
Public on Mar 14, 2007 |
| Title |
NY1DD Sickle Cell Mouse brains, NLX treated, 10mins. SCNLX-I 817 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NY1DD Sickle Cell Mouse brains, NLX treated, 10mins.
|
| Organism |
Mus musculus |
| Characteristics |
Strain:NY1DD Sickle Cell Mice
Gender: Male
Age: 9 wks
Drug: Naloxone for 10 mins.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1) Homogenize brain in 500 microL of TRIzol in a 1.5mL tube with a pestle. Dilute with 500microL.
2)Incubate for 5 mins at RT.
3) Add 200 microL chloroform and vortex. Incubate for 2-3mins and centrifuge at 12000 x g for 15 mins.
4) Transfer upper aqueous portion to a clean tube and precipitate RNA byadding 500 microL of isopropanol. Incubate for 10 mins at RT and centrifuge at 12000 x g for 15 mins.
5) Wash pellet in 75% EtOH and dry pellet for 5-10 mins.
6) Dissolve RNA in 40 microL RNase free water.
|
| Label |
Cy5
|
| Label protocol |
SuperScript Direct labeling system (Invitogen, Cat#: L1015-01) was used and manufacturer's protocol was followed.
|
| |
|
| Channel 2 |
| Source name |
NY1DD Sickle Cell Mouse brains, Saline treated, 10 mins.
|
| Organism |
Mus musculus |
| Characteristics |
Strain:NY1DD Sickle Cell Mice
Gender: Male
Age: 9 wks
Drug: Saline for 10 mins.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1) Homogenize brain in 500 microL of TRIzol in a 1.5mL tube with a pestle. Dilute with 500microL.
2)Incubate for 5 mins at RT.
3) Add 200 microL chloroform and vortex. Incubate for 2-3mins and centrifuge at 12000 x g for 15 mins.
4) Transfer upper aqueous portion to a clean tube and precipitate RNA byadding 500 microL of isopropanol. Incubate for 10 mins at RT and centrifuge at 12000 x g for 15 mins.
5) Wash pellet in 75% EtOH and dry pellet for 5-10 mins.
6) Dissolve RNA in 40 microL RNase free water.
|
| Label |
Cy3
|
| Label protocol |
SuperScript Direct labeling system (Invitogen, Cat#: L1015-01) was used and manufacturer's protocol was followed.
|
| |
|
| |
| Hybridization protocol |
1) Resuspend dried labeled cDNAs in 7.8ul water
2) Add remaining reagents for Hyb cocktail and mix by pipetting.
3) Heat the probe preparation at 95-100 degrees for 2 minutes. Boiling denatures the sample and makes it accessible for hybridization.
4) Snap cool on ice for 1-3 minutes.
5) Pulse the tubes in the centrifuge to pull down condensation
6) Clean lifterslips with soap and water, then Acetone and dry with Kimwipes. Make sure there are no debris on the array before laying down lifterslip. Place lifterslip on array using either fingers or forceps with dull white strips touching the glass. This creates a platform which allows even distribution of the hybridization solution across the array. Slowly pipette the probe under one corner of the slip until the entire array surface is covered.
7) Put 10-20ul 3XSSC at each little divot at the ends of the Hyb chamber. This is to ensure a constant humidity in the chamber during hybridizations. If the 3XSSC is not applied, the array will dry out.
8) Tightly screw down lid of Hyb chamber and carefully place in incubator. Take caution to keep array completely flat during transfer and hybridizations.
9) Allow hybridization to run for at least 5 hours but not more than 16 hours, in the dark at 42C.
|
| Scan protocol |
1) Prepare wash solutions in glass slide dishes
2) Carefully remove array from incubator or water bath, making sure to keep chamber level. Unscrew chamber and remove array.
3) Keep array level and submerge in 2XSSC to get the lifterslip to fall off. Once submerged tilt array and gently dump off coverslip. It may be necessary to lightly swish array under solution to dislodge the slip.
4) Once slip is off and lying on bottom of slide dish, put array in a slide rack and transfer to the first wash solution. Do not allow the chips to dry out at any point during the washes.
5) Wash hybs.
a) 2X SSC, 0.1% SDS (4ml 10%SDS per 400ml) at 37 or 550C for 3 minutes with no agitation
b) 2X SSC, RT Let sit 3 minutes in solution then dip up and down 10 times.
c) 0.2X SSC RT Let sit 3 minutes in solution then dip up and down 10 times
6) Immediately dry array in room temperature table top centrifuge at 500 rpm for 6 min.
|
| Description |
All info provided.
|
| Data processing |
Scanned image was processed using the GenePix Pro (Axon Laboratories) software and RAW values were calculated. The data was then updated in the GeneSpring Software (Agilent Tech.) and lowess normalizations were used on all the raw data.
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| |
|
| Submission date |
Oct 23, 2006 |
| Last update date |
Mar 14, 2007 |
| Contact name |
Ajay Yekkirala |
| Organization name |
University of Minnesota
|
| Street address |
308 Harvard St 8-120
|
| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55112 |
| Country |
USA |
| |
|
| Platform ID |
GPL4464 |
| Series (2) |
| GSE6479 |
Analgesia of naloxone and morphine in wild-type and NY1DD sickle cell mice using BMAP cDNA microarrays |
| GSE6578 |
Analgesia of naloxone in wild-type and NY1DD transgenic mouse model of sickle cell anemia using BMAP cDNA microarrays |
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