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A tomato fruit RNA extraction method that isolates mRNAs encoding secreted and endomembrane associated proteins
Nottingham Arabidopsis Stock Centre (NASC), Nottingham Arabidopsis Stock Centre (NASC), Department of School of Biosciences, University of Nottingham, Loughborough, United Kingdom (affy@arabidopsis.info)
Experiment design (3 hybridizations)
protocol
•RNA Extraction Ex1 •RNA Extraction Ex2 •RNA Extraction Ex2-pellet

This experiment has been imported by PLEXdb from NCBI GEO (GSE17222)

Series_summary:
Background: During the analysis of ripening rela...[complete overview]

Experiment     Expression     Hybridizations & Samples     Quality Control     Compare Treatments     Downloads    

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Experiment Name: A tomato fruit RNA extraction method that isolates mRNAs encoding secreted and endomembrane associated proteins
Accession No: LE16
Microarray: tomato10k
Visibility: public
Experiment Type:
Experiment Factor(s):
protocol
•RNA Extraction Ex1   •RNA Extraction Ex2   •RNA Extraction Ex2-pellet
Quality Control: biological replicates
Treatment summary:
 protocol  # replicates
 RNA Extraction Ex1  1
 RNA Extraction Ex2  1
 RNA Extraction Ex2-pellet  1
Total hybridizations: 3
Description: This experiment has been imported by PLEXdb from NCBI GEO (GSE17222)

Series_summary:
Background: During the analysis of ripening related gene expression in tomato fruit, we observed a bias towards certain classes of messenger RNA based upon the type of buffer used in the initial step of the extraction procedure. We postulated that there was a functional association of the separated transcripts. To test this hypothesis, we carried out extractions from the same tissue sample where only the buffer varied. Transcripts were hybridised to Affymetrix oligo arrays and the data was analysed according to the predicted cellular component of the encoded proteins. Results: The use of an extraction buffer that lacked high levels of caeotropic agents resulted in a reduction of mRNAs encoding proteins that were either secreted or were predicted to be routed through the endomembrane system and hence could have been bound to the endoplasmic reticulum in polyribosomes. Extraction of the cell debris immediately following the initial buffer based extraction and subsequent micro array analysis revealed that the expected transcripts could be recovered. This demonstrated that the buffer was separating the mRNAs based on the cellular component of the encoded proteins. Analysis of selected transcripts by northern hybridisation again supported this theory. Conclusions: Some traditional buffers used for fruit RNA extraction selectively deplete for transcripts encoding proteins that are membrane-associated or secreted. This can be explained if polyribosomes that are bound to the endoplasmic reticulum (ER) are not effectively disrupted when the extraction buffer lacks either detergent or organic solvents. These findings have important implications with respect to experimental bias, as well as opportunities for message enrichment and protein characterisation.

Series_overall_design:
GeneChip analyses were performed to analyse the effect of using different extraction protocols on Solanum lycopersicum pericarp.
Publication: none
Created: 2011-08-01 13:08:39
Last Update: 2011-08-05 09:58:28
Released: 2011-08-05
GEO Accession GSE17222
Submitter: PLEXdb Curator
Name: Nottingham Arabidopsis Stock Centre (NASC)
Institution: Nottingham Arabidopsis Stock Centre (NASC), Department of School of Biosciences, University of Nottingham, Loughborough, United Kingdom
Head of Laboratory: --
email(s):
Homepage:

 
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