World Library  


Add to Book Shelf
Flag as Inappropriate
Email this Book

Plos One : Avoiding Pitfalls of Internal Controls ; Validation of Reference Genes for Analysis by Qrt-pcr and Western Blot Throughout Rat Retinal Development, Volume 7

By Ferreira, Sergio, T.

Click here to view

Book Id: WPLBN0003934285
Format Type: PDF eBook :
File Size:
Reproduction Date: 2015

Title: Plos One : Avoiding Pitfalls of Internal Controls ; Validation of Reference Genes for Analysis by Qrt-pcr and Western Blot Throughout Rat Retinal Development, Volume 7  
Author: Ferreira, Sergio, T.
Volume: Volume 7
Language: English
Subject: Journals, Science, Medical Science
Collections: Periodicals: Journal and Magazine Collection
Historic
Publication Date:
Publisher: Plos

Citation

APA MLA Chicago

Ferreira, S. T. (n.d.). Plos One : Avoiding Pitfalls of Internal Controls ; Validation of Reference Genes for Analysis by Qrt-pcr and Western Blot Throughout Rat Retinal Development, Volume 7. Retrieved from http://worldebookfair.com/


Description
Description : Background : Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. Methodology/Principal Findings : We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (bactin, cyclophilin b, a-tubulin and lamin A/C), while MAPK1 was stably expressed. Conclusion : Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

 

Click To View

Additional Books


  • Plos One : Dampening of Hyperexcitabilit... (by )
  • Plos One : the Dynamics of Sex Ratio Evo... (by )
  • Plos One : Herpes Simplex Virus 1 Icp22 ... (by )
  • Plos One : Highly Efficient Nmr Assignme... (by )
  • Plos One : Autoimmune Gastro-pancreatiti... (by )
  • Plos One : Challenges in the Diagnosis o... (by )
  • Plos One : a Primate Specific Extra Doma... (by )
  • Plos One : Histological Criteria for Enc... (by )
  • Plos One : High-throughput Sequencing an... (by )
  • Plos One : Characterization of the 2009 ... (by )
  • Plos One : Mapping Sub-antarctic Cushion... (by )
  • Plos One : Impacts of an Invasive Non-na... (by )
Scroll Left
Scroll Right

 



Copyright © World Library Foundation. All rights reserved. eBooks from World eBook Fair are sponsored by the World Library Foundation,
a 501c(4) Member's Support Non-Profit Organization, and is NOT affiliated with any governmental agency or department.