Использование Reverse Phase белка массивы (RPPA) изучить Изменение экспрессии белков в отдельных рака почки сотовых
Summary
RPPA позволяет экспрессии белка сотен образцов, напечатанных на нитроцеллюлозные слайды на допрос одновременно, с использованием флуоресцентно меченых антител. Этот метод был применен для изучения влияния препарата неоднородности лечения в течение ясно рак почек.
Abstract
Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.
Protocol
Representative Results
Discussion
Метод RPPA представленные здесь представляет собой высококачественную альтернативу пропускной способности широко используется, но сравнительно низкой пропускной западной техники блот-анализа белка. Метод позволяет сотни образцов, чтобы быть полуколичественным анализируются и сравн…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Работа авторов FCO, DF, JN, DJH и GDS упомянутых выше финансируется за счет главного офиса ученый, номер гранта: ETM37 и при поддержке Cancer Research UK экспериментальный центр медицины Рак. Работа AL финансируется за счет Королевского колледжа хирургов в Эдинбурге Robertson Trust, Мелвилл целевой для ухода и лечения рака и Совет по медицинским исследованиям. IO при поддержке Королевского общества Эдинбурга стипендий Правительства шотландского cofunded Мари Кюри действия и Великобритании Medical Research Council. Авторы хотели бы поблагодарить SCOTRRCC со-заявителей и сотрудников за полезные обсуждения на некоторые из тем, обсуждаемых в данной статье.
Materials
| Name of the reagent | Company | Catalogue number |
| aprotinin | Sigma | A6279 |
| phosphatase inhibitor cocktail 2 | Sigma | P5726 |
| phosphatase inhibitor cocktail 3 | Sigma | P0044 |
| protease inhibitor cocktail | Roche | 11836153001 |
| Triton X-100 | Triton-X | T8787 |
| Li-Cor Odyssey Blocking Buffer | Li-Cor | 927-40000 |
| TissueLyser | Qiagen | 85600 |
| MicroGrid II robotic spotter | Biorobotics | |
| FastFrame’ four bay slide holder | Whatman | 10486001 |
| FAST Slide – 2-Pad | Whatman | 10485317 |
| IRDye 680LT Goat anti-Mouse IgG | Licor | 926-68020 |
| IRDye 800CW Goat anti-Rabbit IgG | Licor | 926-32211 |
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