All Animals had estrous cycles syncronised with 12 animals receiving grade 1 embryo transfers at day 7 of the reproductive cycle, animals were then slaughtered at day 17 of the reproductive cycle and endometrial tissue samples obtained.
Extracted molecule
total RNA
Extraction protocol
Endometrial tissue was homogenised in Qiagen buffer RLT (QIAGEN GmbH, QIAGEN, Hilden, Germany) using FastPrep Lysing Matrix D tubes in a FastPrep instrument (MP Biomedicals, Solon, OH). Total RNA was extracted using a Qiagen RNeasy kit (QIAGEN). All samples were DNase treated using the Ambion DNA-free kit (Ambion, Austin, TX) according to the manufacturer’s instructions. RNA quantity was determined by spectrophotometry in a Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE). RNA integrity was checked using the Agilent 2100 Bioanalyzer with a RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA).
Label
Cy5
Label protocol
One ug of RNA was amplified using the amino Allyl MessageAmp ™ aRNA Kit (Ambion, 2130 Woodward St, Austin TX, 78744) to generate amino allyl modified aRNA for use in microarray hybridisation. aRNA quantity was measured by spectrophotometry using a ND-1000 (Nanodrop Technologies, Wilmington, DE). Five ug of aRNA was then vacuum dried and labelled with Cy3 and Cy5 NHS ester dyes (Amersham Cy3 and Cy5 Mono-Reactive Dye Packs, GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire). Labelled aRNA was then purified on column, quantified and dye coupling efficiency measured using the Nanodrop (Nanodrop Technologies, Wilmington, DE) instrument.
tissue type: intercaruncular and caruncular endometrium reproductive state: pregnant and non-pregnant (cycling)
Treatment protocol
All Animals had estrous cycles syncronised with 12 animals receiving grade 1 embryo transfers at day 7 of the reproductive cycle, animals were then slaughtered at day 17 of the reproductive cycle and endometrial tissue samples obtained.
Extracted molecule
total RNA
Extraction protocol
Endometrial tissue was homogenised in Qiagen buffer RLT (QIAGEN GmbH, QIAGEN, Hilden, Germany) using FastPrep Lysing Matrix D tubes in a FastPrep instrument (MP Biomedicals, Solon, OH). Total RNA was extracted using a Qiagen RNeasy kit (QIAGEN). All samples were DNase treated using the Ambion DNA-free kit (Ambion, Austin, TX) according to the manufacturer’s instructions. RNA quantity was determined by spectrophotometry in a Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE). RNA integrity was checked using the Agilent 2100 Bioanalyzer with a RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA).
Label
Cy3
Label protocol
One ug of RNA was amplified using the amino Allyl MessageAmp ™ aRNA Kit (Ambion, 2130 Woodward St, Austin TX, 78744) to generate amino allyl modified aRNA for use in microarray hybridisation. aRNA quantity was measured by spectrophotometry using a ND-1000 (Nanodrop Technologies, Wilmington, DE). Five ug of aRNA was then vacuum dried and labelled with Cy3 and Cy5 NHS ester dyes (Amersham Cy3 and Cy5 Mono-Reactive Dye Packs, GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire). Labelled aRNA was then purified on column, quantified and dye coupling efficiency measured using the Nanodrop (Nanodrop Technologies, Wilmington, DE) instrument.
Hybridization protocol
825ng of both Cy3 and Cy5 labelled aRNA was used for microarray hybridisation using the Agilent Gene Expression Hybridisation Kit (60-mer oligo microarray protocol version 4.0) (Agilent Technologies, Bioresearch Solutions Unit, 3500 Deer Creek Road, Palo Alto, CA 94034, USA). Dye-labelled, fragmented aRNA was then added to each Agilent 44k 60-mer oligonucleotide microarray, hybridised over night (17 hours), washed and allowed to air dry.
Scan protocol
Arrays were then scanned using the Agilent DNA microarray scanner.
Description
Caruncular_Non-Pregnant_9960
Data processing
Agilent feature extraction software version 7.1 was used to analyse the scanned Agilent microarray. The 44 scanned microarray image files were uploaded to the feature extraction software. Using the design file (015354) the feature extraction software locates features and converts the extracted data from each feature into a quantitative log ratio. The software removes pixel outliers, does statistics on the non outlier pixels, subtracts background from features and flags any outlier features. The software was then used to perform a LOWESS (locally weighted linear regression analysis) dye normalisation and to calculate a p-value for each feature. Data analysis was performed with Genespring GX 7.3.1. (Agilent, Palo Alto, CA, USA). Microarray data were imported into Genespring using Agilent’s two-colour ‘Enhanced FE’ import scenario which included ‘Per Spot: Divide by control channel’ and ‘Per Chip: Normalize to 50th percentile’ normalisation steps. Filters applied to the data to improve the quality of the normalised dataset include; Firstly, filtering ‘on flags’ such that any probes that were not deemed ‘present or marginal’ (according to feature extraction spot quality guidelines) in at least 22 of the 44 arrays were omitted from analysis. Second, probes that did not have a minimum threshold of 300 raw intensity units in at least 22 of 44 arrays were also omitted from analysis. The raw intensity cut off value of 300 was determined based on the calculation of base over proportional (C=a/b) calculation, which is calculated by plotting the standard deviation of normalised values against the control values. The point at which the curve flattens out is where the data measurement becomes reliable or where C (control strength) = a/b (where a = base and b = the proportional coefficient).
