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We note that Hua et al. (2) have also compared various normalization methods for microRNA microarrays, using correlation with PCR results to quantify performance. Although they report print-tip loess normalization as the method that performs best, we find no statistically significant difference between it and other standard methods compared in their study (median normalization, VSN, etc.), as we show in the Tods lace up boots Grey Looking For For Sale Sale 2018 4c8FEckvb
. We have not considered print-tip loess normalization in our analysis as it does not seem applicable to our data; in particular, unlike for Hua et al. (2), a substantial proportion of microRNAs appear to be unexpressed in our samples.

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Biological samples

Primary human ovarian surface epithelial cultures were derived from histologically normal oopherectomy specimens and cultured as described in detail in the Supplementary Material . Snap frozen ovarian cancer tissue specimens corresponding to serous and endometrioid histologies were obtained from the Pacific Ovarian Cancer Research Consortium Repository. All clinical samples in this study were collected under Institutional Review Board-approved protocols.

Explicit details regarding the microarrays are given in the Supplementary Material . Briefly, as shown in Buy Cheap Cheapest Price Quiksilver Carver Crew Shipping Discount Authentic Cheap Popular Fashionable Sale Online Cheap Price Top Quality nV83ObiI
, the arrays had 16 print-tip blocks, each with 238 spots laid out in a 14 × 17 grid. Of these, 672 spots were blank (primarily at block boundaries), 1930 represented nonhuman microRNAs, 904 were human microRNAs and 302 were proprietary probes (miRPlus). Each probe was spotted in duplicate. Two arrays were printed on each slide.

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Figure 1.

Layout of microRNA arrays. There were 16 print-tips, and blocks were laid out on a 4 by 4 grid. The majority of spots do not represent human microRNAs. Of the ones that do, each are spotted in duplicate, so there are ∼450 unique microRNAs in total. Included among the nonhuman microRNAs are 15 spike-in controls, also spotted in duplicate.

PCR validation

For some microRNAs a TaqMan microRNA assay (Applied Biosystems) was used for qRT-PCR. Normalization was done with the RNU24 endogenous control assay. Reverse transcription was carried out with the ABI microRNA Reverse Transcription kit using the manufacturer's; recommended protocol. Real-time PCR was performed on an ABI Prism 7900HT Sequence detection system using 2× Universal PCR Master Mix, no AmpErase UNG. A total of seventeen microRNAs (listed in the Supplementary Material ) were assessed by qRT-PCR.

Spiked-in synthetic nonhuman microRNAs

Fifteen nonhuman microRNAs that did not show cross-hybridization with multiple human tissue RNA samples and that exhibited sufficient signal intensities were used as spike-ins in varying amounts adjusted to maximize the range of the signal they provided, covering the expected span of intensities of biological samples. These were added to samples for both the red and the green channels. See the Supplementary Material for more details, including the identities of the microRNAs used.

Synthetic human microRNA universal reference pool

A synthetic human microRNA universal reference pool was constructed and was used in the green channel in all arrays (we will sometimes refer to this as the reference channel). Briefly, RNA oligonucleotides were synthesized corresponding to 454 microRNAs, of which 56 failed to provide sufficient intensity, for a variety of reasons, leaving 398 that were used on our arrays. Their concentrations were adjusted to provide approximately uniform intensity across spots.

The measured per-spot log 2 intensities may be used to assess the quality of the arrays and spots. The values in the red channel are not suitable for quality assessment, but the green reference channel is useful because it should be the same on all arrays, and the Cy3-labeled (green) synthetic universal reference pool oligos should hybridize to most spots that correspond to human microRNAs. We see an example of this in Figure 2 , which is much like the heatmap, or false color image, that is widely used to convey gene expression data. Rows represent microRNAs, and the columns represent arrays. The microRNAs are ordered by average intensity across arrays, as there is no particularly natural ordering for them. The false color represents intensity, which is scaled to lie between 0 and 1 for each spot; this enhances comparison across arrays at the cost of comparison across spots, which is not of interest.

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