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Figure 4.

Spread-location plot for probes on the red channel. A measure of spread (MAD) across arrays is plotted against a measure of location (median), before and after normalization. Only the spots corresponding to human microRNAs are shown, along with the spike-ins. For successful normalization, we expect the spike-ins to have low variability. By this criterion, all methods are successful; indeed, this holds even for the unnormalized intensities. There is a clear relationship between location and spread in the unnormalized data. The VSN-based methods make the spreads more homogeneous, as expected. The vertical grey line represents the 60th percentile of the median values; we only retain spots with median higher than this value (i.e., the top 40% spots) for further analysis.

Normalization

Our proposed diagnostics, namely, Figures 3 , ,4, 4 , and Supplementary Figure S2 , suggest that all normalization methods perform adequately in our study. Here, we only show results of subsequent analysis using spike-in VSN normalization; results for other methods are given in the Cole Haan Original Grand Penny II Free Shipping Shop Cheap Sale Low Shipping Fee Cheap Sale Affordable Brand New Unisex Sale Online rO4QBr
.

We are interested in microRNAs that have expression roughly in the range of the spike-ins. However, Outlet Locations Popular And Cheap BUGATCHI Novara SlipOn Derby For Cheap Cheap Online Low Shipping Online Clearance Official Site Zrg1Y1
suggests that the majority of the spots have median intensity below this range, and are presumably not expressed. We thus retain for further analysis only those spots corresponding to the top 40% median intensity values. This nonspecific filtering reduces problems associated with multiple testing. Note that the filtering step is by nature somewhat arbitrary, and may need adjustments depending on the purpose of the study; for example, our method may not identify potentially interesting spots that are expressed only in a few samples.

Table 1 presents the list of the top ten differentially expressed microRNAs between the serous and endometrioid subtypes of ovarian cancer. When available, the table is augmented by the result of qRT-PCR (in the form of a log 2 fold change and P -value). The results for other normalization methods, as well as for unnormalized intensities, are provided in the Supplementary Material . The results are largely invariant to the normalization method used; in particular, the same microRNAs appear as the most significantly differentially expressed, with largely similar adjusted P -value.

The top 10 hits using Spike-in VSN normalization

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ND, Not done.

External validation

It should be noted that most of the diagnostic plots we propose are based on the premise that successful normalization methods should reduce variability (across arrays) at spots that correspond to microRNAs that should have constant expression. A good method should additionally ensure that the true signals of interest are also reported accurately. This can be checked by comparing the normalized intensities to expression data obtained using an independent method. The supplementary file qrtpcr.pdf plots average ΔCt values obtained using qRT-PCR against raw and normalized intensities from microarrays for the same set of tissue samples for 17 microRNAs. The agreement can be summarized by correlations, which were fairly strong for most microRNAs, both for unnormalized and normalized intensities. Analysis of variance, described in the Supplementary Material, suggests that the amount of correlation did not differ significantly between normalization methods, especially if we consider the variability inherent in qRT-PCR.

Many of our proposed methods depend on a synthetic universal reference pool and spike-in controls (both available from author M. Tewari upon request). The choice and concentrations of the spike-in controls are specific to the microarray platform used, and will need to be tuned for each array platform. This is possible as long as the relevant platform contains a sufficient number of probes that do not cross-hybridize to human microRNAs. One approach to predict cross-hybridization is to compute similarity with known human microRNA sequences, and although further experiments are required to confirm suitability, an initial set of candidate microRNAs can be obtained using such a score. We used a similarity measure that is essentially the length of the longest common substring, with penalties for mismatches and gaps, to obtain explicit lists of candidate microRNAs for popular commercial platforms, given in Cheap Countdown Package 2018 New Online Comme Des Garçons pitch low spalwart sneakers White Outlet With Credit Card k6sopB0lAh
. Computations are based on mature sequences in miRBase release 12.0 (9) and available information on the contents of each platform. Candidates are sorted by similarity, but are arbitrarily limited to 20 after removing those too similar to candidates already on the list, to ensure that the selected spike-ins do not cross-hybridize with each other. Further details are given in the Supplementary Material .

To what extent is dopaminergic neuronal death attributable to microglial activation as opposed to a direct effect of cyclooxygenase-mediated reactions? Using the activated microglial inhibitor minocycline, Przedborski's group showed that activated microglia contribute to about 20% of the MPTP-induced TH-positive cell death [ 52 ]. The same group also showed 30–40% neuroprotection by the COX-2 inhibitor rofecoxib leaving 74–88% neuronal survival after MPTP injection but failed to show inactivation of microglia by COX-2 inhibition or deficiency [ Newest Sale Online Reliable For Sale PUMA Suede Classic Casual Embossed Sneaker Big Sale Cheap Online Cheap Sale With Credit Card xHLAu
]. The differences between their findings and ours may be due to different experimental settings, procedures, or technical variables, such as using different COX-2 inhibitors, pre-treatment time with the inhibitors, drug/toxin dosages, or unequal ages of mice. In addition, the previous work examined microglial activation at early stages following MPTP administration; thus, it is possible that examination of pathology at later stages is a better indicator of microglial activation. Moreover, a direct neurotoxic role of COX-2 activation cannot explain why COX-2 inhibitors may be protective or toxic in different PD models or systems [ 1 , 53 ]. As Wang et al. suggested, the final effect of inflammation may vary depending on the balance between neurotrophic and neurotoxic factors released by activated microglia in different systems or approaches, and the discrepancy thus may be due rather to an indirect role of COX-2 in neurotoxicity through regulation of inflammation [ MC2 Saint Barth Mens Swimwear camouflage Canvas 2017 US 7 EU 41 US 75 EU 415 US 8 EU 42 Outlet Explore Discount Pay With Paypal Discount Order dYW9929ZDJ
]. The current result could be because of differences in the persistence of neuronal abnormalities or microglial activation.

Figure 9

Schematic flow chart depicting the role of the vicious circle in dopaminergic neurotoxicity. For this study, the initial insult MPP exerts direct dopaminergic neurodegeneration (~30% of the original numbers). Neuronal damage initiates the vicious circle with COX-2 and microglia as the key components feeding oxidative and inflammatory damage to the neurons, which in turn progresses to the secondary damage/death which is coupled to the release of factors that initiate another cycle of oxidative and inflammatory insults. The positive feed back loop may continue until the additional neuronal death (~50% of the remaining of the initial death) combined with the initial loss exceed the amount needed for normal motor control (~80% total loss); thus the PD symptoms, such as postural instability or hypokinesia, occurs. This vicious circle helps explain the chronic and prolong nature of PD progression. It is hypothesized that the blockade of COX-2 activity by selective COX-2 inhibitors or deficiency of COX-2 would attenuate the vicious circle and alleviate dopaminergic neurotoxicity by directly reducing COX-2 free radical production as well as by indirectly decreasing microglial activation and subsequent microglia-mediated damage.

We have not repeated those experiments done by previous groups of investigators, which support our studies. From the previous works by Teismann et al. and others [ 1 , SLIDEON Mules black Buy Cheap Outlet Locations VxOFjc92W
], we have not overlooked another possible interpretation of our results: that microglia may become inactive faster without COX-2. In general, activated immune cells including microglia become inactive over time through normal regulatory processes. Indeed, it has been shown that peak activation of microglia occurs around day 2–3 after MPTP injection, after which microglial activation dissipates [ 1 , 26 ]. Our results show extended activation of microglia two weeks after MPTP injection although this degree of activation is less than that seen during the first few days by other investigators. We also show much lesser amounts of activated microglia in COX-2-deficient or COX-2-inhibited mice. We conclude that COX-2 plays a role in sustaining microglial activation, or that activated microglia may be excluded persistent activation if COX-2 is lacking. In other words, our results showed that COX-2 inhibition or deficiency may be related to decreased microglia activation. With time, activation of microglia declines as COX-2 inhibition helps to reduce the perpetuation of a vicious circle that leads to chronic inflammation and secondary neurodegeneration.

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