Gender-specific alterations in gene expression and loss of liver sexual dimorphism in the long-lived Ames dwarf mice.

Transcription profiling of long-lived Ames dwarf mice investigating the loss of liver sexual dimorphism

Gender-specific alterations in gene expression and loss of liver sexual dimorphims in the long-lived Ames dwarf mice

Affymetrix GeneChip Mouse Expression Array MOE430A [MOE430A]

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Gender-specific alterations in gene expression and loss of liver sexual dimorphism in the long-lived Ames dwarf mice.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4[]

Title: Affymetrix EukGE-WS2v4 Hybridization. Description:

Affymetrix:Protocol:Percentile

Title: Affymetrix CEL Analysis (Percentile). Description:

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-8773

Prop1df /+ female and male heterozygous breeders, purchased from Jackson Labs, were crossed to produce the Prop1+/+ and Prop1df/df male and female 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 in a room with a controlled photoperiod of 12h light:12h darkness 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). Adult animals (3-6 months old) were anesthetized with Isoflurane and killed by cervical dislocation, livers were collected, flash frozen in liquid nitrogen and stored at -70° C.

Transcription profiling of long-lived Ames dwarf mice investigating the loss of liver sexual dimorphism

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