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The Detection Techniques for Autophagy-Associated Cell Death-Related Genes and Proteins: Gene Expression Assay and Immunohistochemistry

  • Melek OzturkEmail author
  • Ozlem Dagdeviren Ozsoylemez
  • Fatma Kaya Dagistanli
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 1854)

Abstract

Autophagy is important in cellular homeostasis for the cell survival mechanism. Deficiency or excess of autophagy is generally related to some of diseases such as cancer and neurodegeneration. Although autophagy is a cell survival mechanism, it can mediate programmed cell death in several conditions. Autophagy-related genes (ATGs) regulate the autophagy and also control the crosstalk with autophagy-associated cell death and apoptosis in some condition. Various methods have been used to detect the marker genes and the proteins involved in these processes. Quantitative real-time PCR (qRT-PCR) method for monitoring the expression of genes involved in autophagy or autophagic cell death is often preferred because of its sensitivity, high efficiency potential, accurate quantification, and high-grade potential automation. The detection of the markers for autophagy-related process by immunohistochemistry in paraffin sections of various patient tissues has become a reliable method for monitoring autophagy. Here, we introduce protocols for detecting autophagy and autophagy-associated cell death in HeLa cells by using gene expression assays qRT-PCR, and also in paraffin-embedded tissue section from human biopsy material by using immunohistochemistry.

Keywords

Autophagy Autophagy-associated cell death Gene expression assay Immunohistochemistry 

Notes

Acknowledgment

This study was supported by the Research Fund of the University of Istanbul, Turkey, Project numbers: 17492, 38781 and 39211.

References

  1. 1.
    Ohsumi Y (2001) Molecular dissection of autophagy: two ubiquitin-like systems. Nat Rev Mol Cell Biol 2:211–216CrossRefPubMedGoogle Scholar
  2. 2.
    Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451:1069–1075CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Lin L, Baehrecke EH (2015) Autophagy, cell death and cancer. Mol Cell Oncol 2:e985913CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Radogna F, Dicato M, Diederich M (2015) Cancer-type specific crosstalk between autophagy, necroptosis and apoptosis as a pharmacological target. Biochem Pharmacol 94:1–11CrossRefPubMedGoogle Scholar
  5. 5.
    Ozsoylemez O, Ozturk M, Sutlupinar N, Kayacan S, Tuncdemir M, Ozcan G (2016) The effects of Colchicum baytopiorum on regulatory genes of apoptotic and autophagic cell death in HeLa cells. Curr Pharm Biotechnol 17:1369–1376CrossRefPubMedGoogle Scholar
  6. 6.
    Dagistanli FK, Ozkaya HM, Kucukyoruk B, Biceroglu H, Metin D, Gazioglu N et al (2016) Preoperative somatostatin analogue treatment might trigger apoptosis and autophagy in tumor tissues of patients with acromegaly: a pilot study. Exp Clin Endocrinol Diabetes. [Epub ahead of print]Google Scholar
  7. 7.
    Rubinstein AD, Kimchi A (2012) Life in the balance a mechanistic view of the crosstalk between autophagy and apoptosis. J Cell Sci 125:5259–5268CrossRefPubMedGoogle Scholar
  8. 8.
    Kang R, Zeh HJ, Lotze MT, Tang D (2011) The Beclin-1 network regulates autophagy and apoptosis. Cell Death Differ 18:571–580CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Mathew R, Karantza-Wadsworth V, White E (2007) Role of autophagy in cancer. Nat Rev Cancer 7:961–967CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Gordy C, He YW (2012) The crosstalk between autophagy and apoptosis: where does this lead? Protein Cell 3:17–27CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ip JC, Pang TC, Glover AR, Soon P, Zhao JT, Clarke S et al (2015) Immunohistochemical validation of overexpressed genes identified by global expression microarrays in adenocortical carcinoma reveals potential predictive and prognostic biomarkers. Oncologist 20:247–256CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Freshney RI (2000) Cell lines culture of animal cells: a manual of basic technique. Wiley-Liss, New YorkGoogle Scholar
  13. 13.
    Pestana EA, Belak S, Diallo A, Crowther JR, Viljoen GJ (2010) Early, rapid and sensitive veterinary molecular diagnostics-real time PCR applications. Springer, New YorkCrossRefGoogle Scholar
  14. 14.
    VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44:619–626CrossRefPubMedGoogle Scholar
  15. 15.
    Yajima T, Yagihashi A, Kameshima H, Kobayashi D, Furuya D, Hirata K et al (1998) Quantitative reverse transcription-PCR assay of the RNA component of human telomerase using the TaqMan fluorogenic detection system. Clin Chem 44:2441–2445PubMedGoogle Scholar
  16. 16.
    Letertre C, Perelle S, Dilasser F, Arar K, Fach P (2003) Evaluation of the performance of LNA and MGB probes in 5'-nuclease PCR assays. Mol Cell Probes 17:307–11Google Scholar
  17. 17.
    Torlakovic EE, Francis G, Garratt J, Gilks B, Hyjek E, Ibrahim M et al (2014) International ad hoc expert panel. Standardization of negative controls in diagnostic immunohistochemistry: recommendations from the international ad hoc expert panel. Appl Immunohistochem Mol Morphol 22:241–252CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kaya-Dagistanli F, Tanriverdi G, Altinok A, Ozyazgan S, Ozturk M (2013) The effects of alpha lipoic acid on liver cell damages and apoptosis induced by polyunsaturated fatty acids. Food Chem Toxicol 53:84–93CrossRefPubMedGoogle Scholar
  19. 19.
    Fraga D, Meulia T, Fenster S (2008) Real-time PCR. Current protocols essential laboratory techniques. Doi:  10.1002/9780470089941.et1003s00
  20. 20.
    Vinod KR, Jones D, Udupa V (2016) A simple and effective heat induced antigen retrieval method. MethodsX 3:315–319CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Melek Ozturk
    • 1
    Email author
  • Ozlem Dagdeviren Ozsoylemez
    • 2
  • Fatma Kaya Dagistanli
    • 1
  1. 1.Medical Biology Department, Cerrahpasa Faculty of MedicineIstanbul UniversityIstanbulTurkey
  2. 2.Department of Radiobiology, Institute of Graduate Studies in Science and EngineeringIstanbul UniversityIstanbulTurkey

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