Exon and junction microarrays detect widespread mouse strain- and sex-bias expression differences. Mus musculus

71 liver RNA samples from three mouse strains - DBA/2J, C57BL/6J and C3H/HeJ - were profiled using a custom-designed microarray monitoring exon and exon-junction expression of 1,020 genes representing 9,406 exons. Gene expression was calculated via two different methods, using the 3'-most exon probe ("3' gene expression profiling") and using all probes associated with the gene ("whole-transcript gene expression profiling"), while exon expression was determined using exon probes and flanking junction probes that spanned across the neighboring exons("exon expression profiling"). Widespread strain and sex influences were detected using a two-way Analysis of Variance (ANOVA) regardless of the profiling method used. However, over 90% of the genes identified in 3' gene expression profiling or whole transcript profiling were identified in exon profiling, along with 75% and 38% more genes, respectively, showing evidence of differential isoform expression. Overall, 55% and 32% of genes, respectively, exhibited strain- and sex-bias differential gene or exon expression. Keywords: Grouped Overall design: Hybridization material was generated through a random-priming amplification of poly[A]+ purified RNA using primers with a random sequence at the 3' end and a fixed motif at the 5' end that was optimized to generate strand-specific cDNA copies of full-length mRNA transcripts [32]. Since the region used for exon and junction probe selection is constrained to a smaller region, more probes contain sequence with suboptimal characteristics (e.g. high GC content or higher homology to other genes). The hybridization of cDNA, rather than cRNA as commonly done, partially mitigates this issue due to higher specificity and lower background levels [24]. Hybridization conditions were as previously described [33]. All 71 samples were hybridized in a two-channel experiment, where one channel was a common reference, generated by pooling all 71 samples in equal mass. Array hybridizations were done in duplicate with fluor reversal to systematically correct for Cy3/Cy5 dye bias. Array images were processed as described to obtain background noise, single channel intensity and associated measurement error estimates [34]. Expression changes between samples and pool were quantified as mean log10(expression ratio), and associated error.

本数据集包含源自3个小鼠品系（DBA/2J、C57BL/6J及C3H/HeJ）的71份肝脏RNA样本，采用定制设计的微阵列进行表达谱分析，该微阵列可监测1020个基因（对应9406个外显子）的外显子及外显子-外显子连接表达水平。
基因表达通过两种不同方法计算：采用3'端最外侧外显子探针的3'基因表达谱分析（3' gene expression profiling），以及采用与基因关联的全部探针的全转录本基因表达谱分析（whole-transcript gene expression profiling）；外显子表达则通过外显子探针与跨越相邻外显子的侧翼外显子-外显子连接探针进行测定，即外显子表达谱分析（exon expression profiling）。
无论采用何种表达谱分析方法，通过双向方差分析（two-way Analysis of Variance, ANOVA）均检测到广泛存在的品系和性别对基因表达的影响。然而，通过3'基因表达谱分析或全转录本基因表达谱分析鉴定的基因中，超过90%可在外显子表达谱分析中被鉴定出，同时还分别额外鉴定出75%和38%的具备差异异构体表达证据的基因。总体而言，分别有55%和32%的基因表现出品系偏好性和性别偏好性的差异基因或外显子表达。
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分组总体实验设计：杂交材料通过对poly(A)+纯化的RNA进行随机引物扩增制备，所用引物的3'端为随机序列，5'端带有优化后的固定基序，可生成全长mRNA转录本的链特异性cDNA（strand-specific cDNA）拷贝[32]。由于外显子和外显子-外显子连接探针的选择区域被限制在较小范围内，更多探针的序列存在次优特性（例如高GC含量或与其他基因较高的同源性）。与常规使用的cRNA杂交不同，采用cDNA杂交可通过更高的特异性和更低的背景水平部分缓解该问题[24]。杂交条件如先前文献所述[33]。所有71份样本均在双通道实验中进行杂交，其中一个通道为通用参考样本，通过将全部71份样本按等质量混合制备得到。芯片杂交实验重复两次，并进行荧光通道反转，以系统校正Cy3/Cy5染料偏差（Cy3/Cy5 dye bias）。芯片图像按前述方法处理，以获取背景噪声、单通道强度及相关测量误差估计[34]。样本与混合参考样本之间的表达变化以平均log₁₀(表达比值)及相关误差进行定量。