Detection of VEGF-A(xxx)b isoforms in human tissues.
Detection of VEGF-A(xxx)b isoforms in human tissues.
复制标题
在人体组织中检测VEGF-A（XXX）B同工型。
DOI：
10.1371/journal.pone.0068399
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Harper SJ
中科院分区：
文献类型：
--
作者：
Bates DO;Mavrou A;Qiu Y;Carter JG;Hamdollah-Zadeh M;Barratt S;Gammons MV;Millar AB;Salmon AH;Oltean S;Harper SJ
血管内皮生长因子-A（VEGF-A）可以通过选择性剪接产生多种亚型。在人类中已经描述了两个同种型家族，以VEGF-A165 a为代表的促血管生成同种型和以VEGF-A165 b为代表的抗血管生成同种型。相同基因的替代同种型的表达水平的实际测定可能会因实验方案而变得复杂，该实验方案有利于一种同种型而不是另一种同种型，并且使用特定的阳性和阴性对照对于解释同种型表达的结果至关重要。在这里，我们解决了一些困难，在实验设计时，调查选择性剪接的VEGF亚型，并讨论使用适当的控制模式。我们证明了为什么使用特定的控制实验可以防止假设VEGF-A165 b不存在，而事实上它是。我们重申并确认先前发表的实验设计方案，这些方案证明了使用阳性对照的重要性。这些包括使用已知的靶序列来显示实验条件适合于q-PCR和RT-PCR的VEGF-A165 b mRNA的PCR扩增，并确保不发生错误引发。我们还提供了证据，证明小鼠中VEGF-A165 b蛋白的检测需要严格控制，以防止通过二抗检测小鼠IgG。我们还表明，人VEGF 165 b蛋白可以从培养的人细胞中免疫沉淀，免疫沉淀VEGF-A的结果是由VEGF-A165 b抗体检测到的蛋白质。这些发现支持以下结论：需要更多关于VEGF-A165 b亚型生物学的信息，并证实了实验设计在此类研究中的重要性，包括使用特定的阳性和阴性对照。
Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.
登录
查看更多内容
是否存在抗血管生成VEGF（VEGFXXXB）？一个警示性的故事。
影响因子：
3.7
作者：
Harris S;Craze M;Newton J;Fisher M;Shima DT;Tozer GM;Kanthou C
通讯作者：
Kanthou C
影响因子：
7.5
作者：
Miller-Kasprzak, E.;Jagodziniski, P. P.
通讯作者：
Jagodziniski, P. P.
DOI：
10.1164/rccm.201112-2191oc
发表时间：
2012-06-01
影响因子：
24.7
作者：
Simpson, Angela;Custovic, Adnan;Martinez, Fernando D.
通讯作者：
Martinez, Fernando D.
影响因子：
8.8
作者：
Pritchard-Jones, R. O.;Dunn, D. B. A.;Qiu, Y.;Varey, A. H. R.;Orlando, A.;Rigby, H.;Harper, S. J.;Bates, D. O.
通讯作者：
Bates, D. O.
影响因子：
3.6
作者：
McFee, Renee M.;Rozell, Timothy G.;Cupp, Andrea S.
通讯作者：
Cupp, Andrea S.