Affymetrix GeneChip Mouse Expression Array MOE430A [MOE430A]

Gene expression profile of long-lived Ames dwarf and Little mice

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Effects of the prop-1 and Ghrhr mutations in gene expression during normal aging in mice.

Transcription profiling of prop-1 and Ghrhr mutations in gene expression during normal aging in mice (Ames dwarf and Little mice)

Effects of the prop-1 and Ghrhr mutations in gene expression during normal aging in mice.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-4903

Prop1 (df/+) females and males heterozygotes breeders, purchased from Jackson Labs, were crossed to produce the +/+ and Prop1(df/df) male mice used in these experiments. We used wild type Prop1(+/+) littermates as controls for the Ames dwarf mice. The animals were weaned at the age of 21-30 days and housed in groups of three to five animals of the same gender. Animals were housed in a room with controlled photoperiod of 12h light:12h darkness (lights on from 0700 to 1900 h) and a temperature of 22+/- 2 C. Animals were given free access to water and pelleted diet (5010 rodent diet, LabDiet, PMI Nutrition International, Brentwood, MO). At the ages of 3, 6, 12 or 24 months, the animals were anesthetized with Isoflurane and sacrificed by cervical dislocation, livers (and other organs used for other studies) were collected, flash frozen in liquid nitrogen and stored at -70 C.

P-MEXP-4904

Little mice breeders were purchased from Jackson Labs. Ghrhr-lit/Ghrhr-lit females and Ghrhr-lit/+ males were crossed to produce the Ghrhr-lit/+ and Ghrhr-lit/Ghrhr-lit male mice used in these experiments. The animals were weaned at the age of 21-30 days and housed in groups of three to five animals of the same gender. Animals were housed in a room with controlled photoperiod of 12h light:12h darkness (lights on from 0700 to 1900 h) and a temperature of 22+/- 2 C. Animals were given free access to water and pelleted diet (5010 rodent diet, LabDiet, PMI Nutrition International, Brentwood, MO). At the ages of 3, 6, 12 or 24 months, the animals were anesthetized with Isoflurane and sacrificed by cervical dislocation, livers (and other organs used for other studies) were collected, flash frozen in liquid nitrogen and stored at -70 C.

P-MEXP-4905

No pooling was used in these experiments. Each array in these experiments corresponds to an individual mouse.

P-MEXP-4906

Total RNA was extracted from frozen livers using the Qiagen RNeasy (Qiagen, Valencia CA, USA) purification kit in accordance with manufacture’s protocols. RNA Quality assessment was performed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).

P-MEXP-4907

The labeling was performed according to the guidelines of the Genechip Expression Analysis Technical manual from Affymetrix (www.affymetrix.com). The Enzo BioArray HighYield RNA Transcript Labeling Kit3 (Affymetrix, P/N 900182) was used for generating labeled cRNA target according to the manufacturer's protocols. Briefly:  Synthesis of Biotin-labeled cRNA using Enzo Bioarray High Yield RNA Transcript Labeling Kit:  •Add the following reagents to PCR tubes, at room temp, in order  Template cDNA /DEPC H2O                       22 ul  10X HY Reaction Buffer (vial 1)                4 ul 10X Biotin-labeled Ribonucleotides (vial 2)    4 ul 10X DTT (vial 3)                        4 ul 10X RNase Inhibitor mix (vial 4)        4 ul 20X T7 RNA Polymerase (vial 5)                2 ul Total vol                               40 ul  •Carefully mix by pipetting, pulse down •Incubate 37° C for 5 hrs. •Use thermomixer to hold temperature and automatically mix every 45 min.  Clean-up of Biotin-labeled cRNA using Qiagen RNeasy Kit:  •Adjust sample volume to 100 ul with H2O,Add 60 ul H2O •Add 350 ul RLT buffer containing βME, and mix thoroughly with pipetting •Ad 250 ul 96-100% ETOH, mix by pipetting •Apply sample to RNeasy column, do quick spin discard flow through and tube •Transfer column to new 2 ml collection tube. •Pipet 500 ul RPE Buffer onto column, quick spin, Discard flow through and reuse tube •Add another 500 ul RPE Buffer to the column •Spin 2 min to dry column •Toss flow through and spin again in same tube for 1 min to dry •Transfer column to a new 1.5 ml tube •Elute 2 times with 30 ul H2O each, for 60 ul total •Dry sample down to pellet  •Resuspend pellet in 33ul H2O  Fragmenting the cRNA:  • Combine 15 ug cRNA, H2O, and 5X buffer in 1.5 ml tubes • Incubate 94° C for 35 min, → ice.  

P-MEXP-4908

Hybridization was performed according to Affymetrix protocols for Genechips expression arrays. briefly:  Prepare master mix of hyb solution: Fragmented cRNA                  40 ul  DEPC H2O                         84 ul  Control oligo B2                  5 ul  20X Eukaryotic Hyb controls*     15 ul  Herring sperm DNA, (10 mg/ml)     3 ul  Acetylated BSA, 50 mg/ml)         3 ul 2X Hybridization Buffer         150 ul Final vol:                      300 ul  •Add 260 ul hyb solution to each sample •Heat samples/hyb cocktails to 99° C for 5 min. •Meanwhile, wet the array with 200 ul  1X Hyb buffer Incubate array with buffer at 45° C in hyb oven, with rotation, (60 rpm,) for 10 min minimum •Transfer samples/ hyb cocktails to 45° C for 5 min •Spin samples, max speed, 5 min, room temp. •Remove 1X hyb buffer from cassettes •Pipet 200 ul off top of sample, avoiding any pelleted debris,   and load into cassettes •Cover septa with tough tags to avoid evaporation of sample •Incubate 16 hr, 45° C, at 60 rpm. 

P-MEXP-4910

P-MEXP-4912

No treatment was given to any of the animals used in these experiments.

Transcription profiling of prop-1 and Ghrhr mutations in gene expression during normal aging in mice (Ames dwarf and Little mice)

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