strain: C57BL/6J wild type age: 6 weeks group: control tissue: liver
Treatment protocol
3-4 day old neonate mice were orally infected by gavage with 2x105 G. lamblia ATCC 5058 (assemblage B, strain GS, clone H7) trophozoites.
Growth protocol
G. lamblia was cultured in vitro in medium containing (per liter): Trypticase Peptone 20g, Yeast Extract 10g, D (+) Glucose 10g, NaCl 2g, K2HPO4 1g, KH2PO4 0.6g, L-Cysteine 2g, L(+)Ascorbic Acid 0.2g, Ammonium Citrate 22.8mg, Bile 0.5g, Ampicillin (50ug/ml) 50mg, Kanamycin (100ug/ml) 100mg supplemented with 100ml (10%) of heat Inactivated bovine serum (not fetal bovine serum) and adjusted to pH to 7-7.1 using 5M NaOH and filter sterilzed (0.22um) using a vacuum filter. Prior to infection, parasites were put on ice and parasites released from the flask bottom were counted (Neubauer), washed (2000rpm for 5-7 minutes) and adjusted.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated with Trizol according to the mansufacturer's protocol and stored at -80°C.
Label
Cy3
Label protocol
150 ng of total RNA were used as input. Synthesis of Cy3-labeled cRNA was performed in ¾ reaction volumes with the ‘Low Input Quick Amp Labeling Kit One-Color’ (#5190-2305, Agilent Technologies) according to the manufacturer’s recommendations.
Hybridization protocol
cRNA fragmentation, hybridization and washing steps were carried-out exactly as recommended in the ‘One-Color Microarray-Based Gene Expression Analysis Protocol V6.7’.
Scan protocol
Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel resolution 3 µm, bit depth 20).
Description
M5709 Gene expression in total liver tissue of infected and of age-matched control animals
Data processing
Data extraction was performed with the ‘Feature Extraction Software V10.7.3.1’ by using the recommended default extraction protocol file ‘GE1_107_Sep09.xml’. Measurements of on-chip replicates were averaged using the geometric mean of processed intensity values of the green channel, ‘gProcessedSignal’ (gPS) to retrieve one resulting value per unique non-control probe. Single Features were excluded from averaging, if they i) were manually flagged, ii) were identified as Outliers by the Feature Extraction Software, iii) lay outside the interval of ‘1.42 x interquartile range‘ regarding the normalized gPS distribution of the respective on-chip replicate population, or, iv) showed a coefficient of variation of pixel intensities per Feature that exceeded 0.5. Averaged gPS values were normalized by global linear scaling. For this, all gPS values of one sample were multiplied by an array-specific scaling factor. This factor was calculated by dividing a ‘reference 75th Percentile value’ (set as 1500 for the whole series) by the 75th Percentile value of the particular Microarray to be scaled (‘Array i’ in the formula shown below). Accordingly, normalized gPS values for all samples (microarray data sets) were calculated by the following formula: normalized gPSArray i = gPSArray i x (1500 / 75th PercentileArray i) A lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 15 normalized gPS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 15. Normalized processed signal intensity values (non-log), averaged across on-chip replicates / quantuplicate measurements. Measurements of control features were removed.
