Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data
Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data
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DOI：
10.1186/1471-2199-8-62
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发表时间：
2007-07-25
影响因子：
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通讯作者：
Day, Philip Jr
中科院分区：
文献类型：
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作者：
Maccoux, Lindsey J.;Clements, Dylan N.;Day, Philip Jr
背景资料：真实的实时逆转录酶定量聚合酶链反应（真实的实时RTqPCR）是生物系统中基因表达的最准确测量。不同样本的比较需要通过一个称为归一化的过程进行数据转换。参考或管家基因是基于样品间的组成型表达选择的候选基因，并且允许定量基因表达的变化。目前，还没有鉴定出任何生物体的参考基因，其对于在不同组织类型或疾病情况下使用是普遍最佳的。我们使用微阵列数据来鉴定从正常和骨关节炎犬关节组织（骨、韧带、软骨、滑膜和脂肪）中分离的总RNA产生的新参考基因。设计RTqPCR测定并应用于每种不同的关节组织。参考基因表达的稳定性和排名进行了比较，使用三种不同的数学algorithm.Results：12个新的潜在的参考基因从微阵列数据中确定。一个基因（线粒体核糖体蛋白S7 [MRPS 7]）在所有5个评价的关节组织中稳定表达。一种基因HIRA相互作用蛋白5同种型2 [HIRP 5]）在四种评价的组织中稳定表达。一个常用的参考基因甘油醛-3-磷酸脱氢酶（GAPDH）没有稳定表达的任何组织评价。在Bestkeeper（（R））c和geNorm之间观察到参考基因的等级排序之间最一致的一致性，尽管每种方法倾向于同意每种组织的最稳定表达的基因和最不稳定表达的基因的身份。新的参考基因，确定使用微阵列数据规范化，在传统的方式是更稳定的微阵列数据规范化，通过使用一个真实的-时间RT-qPCRmethodology.Conclusion：微阵列数据规范化，可以使用一个简单的分步程序，以确定新的参考基因，其中一些将表现出良好的措施的稳定性。线粒体核糖体蛋白S7是一个新的参考基因，值得在犬的其他组织和疾病中进行研究。当不存在共调节时，参考基因稳定性评估的不同方法通常会同意最稳定和最不稳定表达的基因。
Background: Real- time reverse transcriptase quantitative polymerase chain reaction (real- time RTqPCR) is the most accurate measure of gene expression in biological systems. The comparison of different samples requires the transformation of data through a process called normalisation. Reference or housekeeping genes are candidate genes which are selected on the basis of constitutive expression across samples, and allow the quantification of changes in gene expression. At present, no reference gene has been identified for any organism which is universally optimal for use across different tissue types or disease situations. We used microarray data to identify new reference genes generated from total RNA isolated from normal and osteoarthritic canine articular tissues ( bone, ligament, cartilage, synovium and fat). RTqPCR assays were designed and applied to each different articular tissue. Reference gene expression stability and ranking was compared using three different mathematical algorithms.Results: Twelve new potential reference genes were identified from microarray data. One gene ( mitochondrial ribosomal protein S7 [ MRPS7]) was stably expressed in all five of the articular tissues evaluated. One gene HIRA interacting protein 5 isoform 2 [ HIRP5]) was stably expressed in four of the tissues evaluated. A commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) was not stably expressed in any of the tissues evaluated. Most consistent agreement between rank ordering of reference genes was observed between Bestkeeper((R)) c and geNorm, although each method tended to agree on the identity of the most stably expressed genes and the least stably expressed genes for each tissue. New reference genes identified using microarray data normalised in a conventional manner were more stable than those identified by microarray data normalised by using a real- time RT-qPCR methodology.Conclusion: Microarray data normalised by a conventional manner can be filtered using a simple stepwise procedure to identify new reference genes, some of which will demonstrate good measures of stability. Mitochondrial ribosomal protein S7 is a new reference gene worthy of investigation in other canine tissues and diseases. Different methods of reference gene stability assessment will generally agree on the most and least stably expressed genes, when co-regulation is not present.