रिवर्स चरण प्रोटीन (RPPA) का उपयोग करने के लिए व्यक्तिगत गुर्दे सेल कैंसर के भीतर प्रोटीन अभिव्यक्ति रूपांतर का अन्वेषण
Summary
RPPA नमूने के सैकड़ों, nitrocellulose स्लाइड पर मुद्रित करने के लिए एक साथ पूछताछ की, fluorescently लेबल एंटीबॉडी का उपयोग प्रोटीन की अभिव्यक्ति के लिए सक्षम बनाता है. इस तकनीक स्पष्ट सेल गुर्दे कार्सिनोमा के भीतर दवा उपचार विविधता के प्रभाव का अध्ययन करने के लिए लागू किया गया है.
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
RPPA यहाँ प्रस्तुत पद्धति एक उच्च throughput प्रोटीन विश्लेषण के बारे में व्यापक रूप से इस्तेमाल किया है, लेकिन अपेक्षाकृत कम throughput वेस्टर्न ब्लॉट तकनीक विकल्प का प्रतिनिधित्व करता है. विधि नमूने के सैकड़ों अर्…
Disclosures
The authors have nothing to disclose.
Acknowledgements
FCO, DF, जे.एन., DJH और GDS लेखकों के काम का उल्लेख ऊपर मुख्य वैज्ञानिक कार्यालय, अनुदान संख्या द्वारा वित्त पोषित है: ETM37 और कैंसर रिसर्च यूके के प्रयोगात्मक कैंसर चिकित्सा केंद्र द्वारा समर्थित है. अल का काम एडिनबर्ग Robertson ट्रस्ट, देखभाल और कैंसर और चिकित्सा अनुसंधान परिषद के इलाज के लिए Melville ट्रस्ट के सर्जन के रॉयल कॉलेज द्वारा वित्त पोषित है. कब मैरी क्यूरी प्रक्रिया और ब्रिटेन चिकित्सा अनुसंधान परिषद द्वारा cofunded एडिनबर्ग स्कॉटिश सरकार फैलोशिप के रॉयल सोसाइटी के द्वारा समर्थित है. लेखकों के इस पत्र में चर्चा विषयों में से कुछ पर उनके उपयोगी विचार – विमर्श के लिए SCOTRRCC सह आवेदकों और सहयोगियों का शुक्रिया अदा करना चाहते हैं.
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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