Compared differentially express genes by sex in mouse for the following tissues: hypothalamus, liver, kidney, ovaries and testis (3 biological x 2 technical replicates for each tissues/sex). We used Affymetrix MOE430A Genechip arrays.

Transcription profiling of hypothalamus, liver, kidney, ovaries and testis from male and female humans and mice

Major Molecular Differences Between Mammalian Sexes are involved in Drug Metabolism and Renal Function

Affymetrix GeneChip Mouse Expression Array MOE430A [MOE430A]

Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

Major molecular differences between mammalian sexes are involved in drug metabolism and renal function

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Compared differentially express genes by sex in mouse for the following tissues: hypothalamus, liver, kidney, ovaries and testis (3 biological x 2 technical replicates for each tissues/sex). We used Affymetrix MOE430A Genechip arrays.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4[]

Title: Affymetrix EukGE-WS2v4 Hybridization. Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4_450[]

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-3957

P-MEXP-4029

No treatment all animals are healthy adults

P-MEXP-4030

For each biological sample,  the mRNA from 10 animals (all male or all female) were pooled. Three different pools of 10 animals each were prepared for each sex and tissue.

P-MEXP-4031

Triple selected polyA mRNA was prepared for each tissue using three rounds of  Polydt beads.

P-MEXP-4032

All samples were labeled using standard affymetrix protocols. Briefly, Double stranded cDNA is prepared with a T7 promoter. The DScDNA is then in vitro transcribed with biotin nucleotides. The biotin aptamer was furhter targeted with Cy3 labeled streptavidin.  Full Description: Preparation of labeled cRNA for hybridization onto Affymetrix GeneChips will follow the recommended  Affymetrix protocol.  Double stranded cDNA will be synthesized from 6 ?g of total RNA by using a Superscript  Choice System (InVitrogen), with an HPLC-purified oligo (dT) 24 primer containing a T7 RNA polymerase  promoter sequence at it’s 5’-end (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3’),  (Genset Corp., La Jolla, CA). The second cDNA strand will be synthesized by using E. Coli DNA polymerase I,  RNase H and DNA ligase.  After second strand synthesis, reactions will be extracted with  phenol/chloroform/isoamyl alcohol. The cDNA will be precipitated with ethanol and resuspended in RNase-free  water.  Labeled cRNA will be generated from cDNA by in vitro transcription using a Bioarray High Yield RNA  Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY) following the manufacturer’s instructions and  incorporating biotinylated CTP and UTP.  Biotin-labeled cRNA will be purified using an RNeasy column prior to  fragmenting to a size of 35-200 bases by incubating at 94 0 C for 35 minutes in fragmentation buffer (40 mM  Tris-acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate).  Integrity of cDNA, cRNA and  fragmented cRNA will all be assessed by running aliquots on the Bioanalyzer prior to proceeding further.

P-MEXP-4033

Preparation of labeled cRNA for hybridization onto Affymetrix GeneChips will follow the recommended  Affymetrix protocol.  Double stranded cDNA will be synthesized from 6 ?g of total RNA by using a Superscript  Choice System (InVitrogen), with an HPLC-purified oligo (dT) 24 primer containing a T7 RNA polymerase  promoter sequence at it’s 5’-end (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3’),  (Genset Corp., La Jolla, CA). The second cDNA strand will be synthesized by using E. Coli DNA polymerase I,  RNase H and DNA ligase.  After second strand synthesis, reactions will be extracted with  phenol/chloroform/isoamyl alcohol. The cDNA will be precipitated with ethanol and resuspended in RNase-free  water.  Labeled cRNA will be generated from cDNA by in vitro transcription using a Bioarray High Yield RNA  Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY) following the manufacturer’s instructions and  incorporating biotinylated CTP and UTP.  Biotin-labeled cRNA will be purified using an RNeasy column prior to  fragmenting to a size of 35-200 bases by incubating at 94 0 C for 35 minutes in fragmentation buffer (40 mM  Tris-acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate).  Integrity of cDNA, cRNA and  fragmented cRNA will all be assessed by running aliquots on the Bioanalyzer prior to proceeding further.    We will take advantage of the relatively inexpensive Test-3 Gene Array to assess the quality of the  biotin-labeled cRNA prior to using the considerably more expensive U133 arrays. Array hybridization buffer  (100 mM MES, 1M [Na +], 20 mM EDTA, 0.01 % Tween 20) will be used to prehybridize the Test-3 Gene array  for 10-15 minutes at 45 0C. The prehybridized solution will be removed and replaced with 80 ?l of hybridization  mixture containing hybridization buffer, fragmented cRNA (0.05?g/?l) and herring sperm DNA (0.1 mg/ml;  Promega).  Also included in the hybridization buffer are acetylated BSA (0.5 mg/ml, Invitrogen) and four control  bacterial and phage cRNA (1.5 pM BioB, 5 pM BIO C, 25 pM BioD, and 100 pM Cre) samples to serve as  internal controls for hybridization efficiency.  The arrays will be hybridized for 16 h at 45 0 in a rotisserie oven.   After hybridization, arrays will be washed using the Affymetrix fluidics station and stained with streptavidinphycoerythrin (10 ?g/ml, Molecular Probes) according to the Affymetrix technical manual. Washed arrays will  be scanned on an Agilent GeneArray Scanner.   

P-MEXP-4034

  Scanned output files will be visually inspected for hybridization artifacts and then analyzed by using  Affymetrix Microarray 5.0 (MAS) software. Arrays will be scaled to an average intensity of 500 and analyzed  independently. The quality of the data will be evaluated by checking the following quality control parameters:    a. Presence of spiked control cRNAs: Bio-B, C, D and CRE serve as positive controls for hybridization and are  spiked at different concentrations as follows: BioB: 1.5 pm, BioC: 5 pM, BioD: 25 pM, CRE: 100 pM. The data  will be considered acceptable if the hybridization intensities for these controls are present in increasing  amounts, B being the least and CRE the highest.     b. Background values: The background value provides a measure of the signal intensity resulting from autofluorescence of the array surface and nonspecific binding of target or stain molecules. The background values  for all the arrays in one experiment should be within +25% of the overall average background; otherwise, this  will introduce additional error. Non-specific binding causes low signal to noise ratio, which may result in genes  for transcripts present at very low levels in the sample being incorrectly called as absent. Thus, high  background noise creates an overall loss of sensitivity in the experiment.    c. Q value. Noise (Q value) results from small variations in the digitized signal observed by the scanner as it  samples the probe array’s surface. It is measured by examining pixel-to-pixel variations in signal intensities.  The noise value for all the arrays in one experiment should be very similar to each other.   

P-MEXP-4035

For each array, the Intensity units from MAS5.0 will be scaled to an average intensity of 500 and analyzed  independently.

P-MEXP-4056

For each biological sample,  the mRNA from a single deceased adult human (either male or female) between the ages of 70-85. The experiment was performed for 7 different males and 5 different females. 

Transcription profiling of hypothalamus, liver, kidney, ovaries and testis from male and female humans and mice

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