Home > Database > Microarray Protocol

Microarray Protocol

Platforms

Underlying the database is a series of gene chip expression profiles for mutant (M) and wildtype (WT) cerebellum across multiple developmental ages (from E12 to E19 in the prenatal and from P0 to P56 in the adult).  These profiles have been obtained using the Affymetrix U74Av2, M430A&B, M430v2, and Illumina Mouse WG-6 v1.1 & v2.0 gene expression profile platforms.

Tissue Collection

Cerebellar tissue from each embryo or postnatal mouse was quickly isolated in ice-cold PBS and frozen in liquid nitrogen.  The tissues from each line were pooled (minimum of three individuals per sample).  Care is taken to avoid thawing prior to homogenization of the tissue in RNASTA60 (Tel-Test Inc.).  The tissue is disrupted by passage through a 23g syringe needle and then passed over a Qiashredder column to ensure complete disruption.  The homogenate is cleared by centrifugation and the RNA precipitated from the supernatant with isopropanol.  The RNA is quantified and QC performed with an Agilent Bioanalyzer 2100 to determine RNA integrity from these tissue pools.  A minimum of 5ug of total RNA is used for generating cRNA for hybridizations (typically 10-15ug, except for very young embryonic ages).  Biotinylated cRNA probes were generated from total RNA, tested for quality and hybridized on a gene chip.  Each chip serves as an independent hybridization experiment and three to four pooled samples were analyzed for each age.

Normalization

Once the microarray chips have been produced, they are processed by software packages designed to extract the expression values.

Affymetrix

The data presented here were processed using Microarray Analysis Suite (MAS) 5.0 software to generate expression level data for each transcript. The mean probe set MAS 5.0 expression level signal is transformed by log2 (X+1) and scaled to a mean signal level near 8. The log2 transformed chip data sets show more nearly normal distributions in our preliminary results. All expression level data in the database (under the column heading Exp. Lvl or Exp. Level) are presented as log2 and thus, a difference of 1.0 units represents a two-fold difference in expression. A minimum of three independent microarray hybridization experiments we done for each group, allowing simple statistical comparisons expression levels from mutant and their respective WT controls using a Students t-test. The p-values for the statistical comparisons are presented (Pvalue) on each gene detail page for WT-Mutant comparisons or comparisons of expression levels at different ages.

Project funded by funded by NIH Grant HD052472. Please view our funding & data sharing policy
Contact Webmaster