Gene expression analysis in heifer luteal tissue by interspecies microarray analysis. Post-pubertal heifer fed diet containing endophyte free fescue seed. Heifer was slaughtered at a local abattoir 11 days after ovulation and used for this study. Ovaries were collected and shipped at room temperature to the laboratory. Approximately 3 mm3 samples of luteal tissue were placed in cryovials and frozen at -80oC until processed. Two samples were ground to a fine powder in liquid N and total RNA was extracted using the RNeasy® Mini Kit (Qiagen, Valencia, CA). Quality and quantity of RNA was determined by gel electrophoresis and spectrophotometry. Five µg of total RNA was converted to cDNA and then biotin-labeled cRNA by linear amplification (CodeLink® Expression Bioarrays, Amersham Biosciences Corp, Piscataway, NJ, USA). Ten micrograms of labeled cRNA were hybridized to CodeLink UniSet Rat I Bioarray (Amersham Biosciences Corp, Piscataway, NJ, USA) by the Amersham Biosciences Facility.
Endophyte-infected diet alters gene expression in heifer luteal tissue
Data table header descriptions
ID_REF
Raw_Intensity
Intensity is defined as the average of the pixel signals in the area of interest minus the scaled 5-pixel median background.
VALUE
Normalized_Intensity. The intensities are median-normalized by calculating the median of all spots, minus the control probes and blanks, and dividing each spot intensity by the median value.
Above_Threshold
After median-normalization, the negative control threshold is calculated using a set of pre-determined negative controls. Negative control threshold = (20% trim-mean of negative controls) + 3 standard deviations using the trim-mean set.
Quality_Flag
Good - No flags associated with this particular spot; Empty - Spots near a background level where the mean background plus one standard deviation is greater than the signal mean; MSR - Data points not provided (blank cell in excel worksheet) based upon quality control of manufacturing processes. Each array is scanned post-dispense and issues such as misfiring are labeled as MSR; Poor - Spots identified based upon characteristics such as spot morphology or large interference in signal (such as a speck of dust); Negative - Spots with a signal mean less than the background, intensity value is a negative number.
