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Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels

Agardh, Elisabet LU ; Gustavsson, Carin LU ; Hagert, Per LU ; Bengtsson, Marie LU and Agardh, Carl-David LU (2006) In Metabolism, Clinical and Experimental 55(2). p.168-174
Abstract

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values... (More)

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Animals, Catalase/analysis, Cyclophilins/analysis, Enzymes/chemistry, Female, Glutamate-Cysteine Ligase/analysis, Glutathione Peroxidase/analysis, Immunoblotting, Peptidylprolyl Isomerase/analysis, RNA/chemistry, Rats, Rats, Wistar, Retina/chemistry, Reverse Transcriptase Polymerase Chain Reaction, Superoxide Dismutase/analysis
in
Metabolism, Clinical and Experimental
volume
55
issue
2
pages
7 pages
publisher
Elsevier
external identifiers
  • wos:000235268000004
  • pmid:16423622
  • scopus:30744435128
  • pmid:16423622
ISSN
1532-8600
DOI
10.1016/j.metabol.2005.08.009
language
English
LU publication?
yes
id
18051944-c3d5-4c33-8a9a-643b16e9844c (old id 150225)
date added to LUP
2016-04-01 15:40:18
date last changed
2021-04-19 08:44:36
@article{18051944-c3d5-4c33-8a9a-643b16e9844c,
  abstract     = {<p>The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.</p>},
  author       = {Agardh, Elisabet and Gustavsson, Carin and Hagert, Per and Bengtsson, Marie and Agardh, Carl-David},
  issn         = {1532-8600},
  language     = {eng},
  number       = {2},
  pages        = {168--174},
  publisher    = {Elsevier},
  series       = {Metabolism, Clinical and Experimental},
  title        = {Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels},
  url          = {http://dx.doi.org/10.1016/j.metabol.2005.08.009},
  doi          = {10.1016/j.metabol.2005.08.009},
  volume       = {55},
  year         = {2006},
}