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Differential Gene Expression Analysis of Pure Cell Populations from Frozen UterineTissue Using Laser Capture Microdissection (LCM) and cDNA Microarrays

ed the assembly such that the extraction buffer completely covered the cap surface.
c) Incubated at 42oC for 30min.
d) Performed RNA binding onto the silica column followed by washing as prescribed in the PicoPure RNA Isolation kit user guide.
e) Eluted the RNA in 11 l elution buffer following the PicoPure protocol.

2. This RNA sample was directly used for amplification using the RiboAmp RNA Amplification Kit (Arcturus, Cat. # KIT0201). After two rounds of amplification, the aRNA was run on an agarose gel, and appeared as a smear, ranging from under 200 bases to over 600 bases in length (Figure 1B). The quality and quantity of aRNA after amplification were further examined by bioanalyzer (Agilent Technologies, Palo Alto, CA) (Figure 1C). Eight to ten micrograms (8-10 g) of aRNA was obtained from CapSure caps filled with microdissected cells, enough for quadruple hybridizations for each sample, using 2 g of aRNA per hybridization.




MICROARRAY ANALYSIS USING THE aRNA
1. The aRNA was subjected to cDNA microarray analysis. Amplified RNA was labeled with Cy3 or Cy5 by direct incorporation of Cy3-dUTP or Cy5 dUTP in the reverse transcription reaction using random hexamer as primer. Labeled aRNA from IM and INTER sites were co-hybridized to TwinChip Mouse 6K cDNA chips (Digital Genomics Inc., Seoul, Korea) at 58C for 16 hours, then washed.

2. Four hybridizations were performed for each sample, including dye-swap, and microarrays were scanned using ScanArray Lite (Perkin Elmer, Billerica, MA). Scanned images were analyzed with GenePix software version 3.0 (Axon Instruments, Union City, CA) to obtain gene expression ratios. Data was then analyzed using Significance Analysis of Microarrays software program (SAM,
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