Identification of suitable reference genes for gene expression studies in tendons from patients with rotator cuff tear.
Identification of suitable reference genes for gene expression studies in tendons from patients with rotator cuff tear.
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DOI：
10.1371/journal.pone.0118821
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Cohen M
中科院分区：
文献类型：
--
作者：
Leal MF;Belangero PS;Figueiredo EA;Cohen C;Loyola LC;Andreoli CV;Smith MC;de Castro Pochini A;Ejnisman B;Cohen M
肩袖撕裂是肩关节功能障碍最常见的原因之一。基因表达分析可能是了解肌腱撕裂和断端愈合失败的有用工具，逆转录定量聚合酶链反应（RT-qPCR）已成为此类研究的有效方法。然而，这种技术需要使用合适的参考基因进行数据标准化。在这里，我们评估了六个参考基因（18 S，ACTB，B2 M，GAPDH，HPRT 1和TBP）的适用性，使用来自28个肌腱撕裂（3个肌腱区域）和8个对照（2个肌腱区域）的肩袖肌腱样本;对于撕裂患者，我们评估了断裂和非断裂肌腱样本。候选参考基因的稳定性使用Normalware、geNorm、BestKeeper和DataAssist软件包确定。总体而言，HPRT 1是最好的单个参考基因，HPRT 1 +TBP组成最好的参考基因对，HPRT 1 +TBP+ACTB组成最好的三个参考基因，来自不同组的分析，包括所有组织样品的同时分析。为了确定参考基因的最佳组合，我们评估了COL 1A 1和COL 3A 1的表达，并且在大多数分析中使用2、3或4个参考基因时没有观察到明显差异。然而，只有当HPRT 1 +TBP+ B2 M和HPRT 1 +TBP标准化时，患者的破裂和非破裂（后上级区域）肌腱之间的COL 3A 1表达才有差异。另一方面，使用最佳的三个参考基因（HPRT 1 +TBP+ACTB）和4个参考基因的这两组之间的比较没有揭示COL 3A 1表达的显著差异。因此，使用合适的参考基因进行可靠的基因表达评估RT-qPCR应考虑肌腱样本的类型进行研究。HPRT 1 +TBP+ACTB似乎是用于分析涉及肩袖撕裂个体的不同肌腱样本的参考基因的最佳组合。
Rotator cuff tear is one of the most common causes of shoulder dysfunction. Gene expression analysis may be a useful tool for understanding tendon tears and the failure of cuff healing, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluate the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1 and TBP) using samples from the rotator cuff tendons of 28 individuals with tendon tears (3 tendons regions) and 8 controls (2 tendon regions); for the tear patients, we evaluated ruptured and non-ruptured tendon samples. The stability of the candidate reference genes was determined using the NormFinder, geNorm, BestKeeper and DataAssist software packages. Overall, HPRT1 was the best single reference gene, and HPRT1+TBP composed the best pair and HPRT1+TBP+ACTB composed the best trio of reference genes from the analysis of different groups, including the simultaneous analysis of all tissue samples. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1 and COL3A1, and no obvious differences were observed when using 2, 3 or 4 reference genes for most of the analyses. However, COL3A1 expression differed between ruptured and non-ruptured (posterior superior region) tendons of patients only when normalized by HPRT1+TBP+B2M and HPRT1+TBP. On the other hand, the comparison between these two groups using the best trio of reference genes (HPRT1+TBP+ACTB) and 4 reference genes did not revealed a significant difference in COL3A1 expression. Consequently, the use of suitable reference genes for a reliable gene expression evaluation by RT-qPCR should consider the type of tendon samples investigated. HPRT1+TBP+ACTB seems to be the best combination of reference genes for the analysis of involving different tendon samples of individuals with rotator cuff tears.
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DOI：
10.1097/01.blo.0000037442.23683.35
发表时间：
2003-02-01
影响因子：
4.2
作者：
Lo, IKY;Marchuk, L;Frank, CB
通讯作者：
Frank, CB
影响因子：
3.7
作者：
de Jonge HJ;Fehrmann RS;de Bont ES;Hofstra RM;Gerbens F;Kamps WA;de Vries EG;van der Zee AG;te Meerman GJ;ter Elst A
通讯作者：
ter Elst A
影响因子：
2.8
作者：
Eriksen, HA;Pajala, A;Risteli, J
通讯作者：
Risteli, J
影响因子：
3.7
作者：
Fu, Jie;Bian, Lihong;Song, Haifeng
通讯作者：
Song, Haifeng
影响因子：
2.9
作者：
Bramono, DS;Richmond, JC;Altman, GH
通讯作者：
Altman, GH