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Bioscience Methods
BM 2011, Vo.2, No.6
http://bm.sophiapublisher.com
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Research Article Open Access
Transcriptome Subtractive Hybridization and Its Reliability Validated by an
E. coli
Model
Yijing Ma , Zhiping Hong , Qin Wu , Xinling Yao
School of Life Science, Ningxia University, Yinchuan, 750021, P.R., China
Corresponding author email: chinanoahl@163.com;
Author
Bioscience Methods 2011, Vol.2 No.6 DOI:10.5376/bm.2011.02.0006
Received: 22 Nov., 2011
Accepted: 28 Dec., 2011
Published: 30 Dec., 2011
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article as:
Ma et al., 2011, Transcriptome Subtractive Hybridization and its Reliability Validated by an
E. coli
Model, Bioscience Methods, Vol.2 No.6
(doi:10.5376/bm.2011.02.0006)
Abstract
In the application of subtractive hybridization methods to identify differentially expressed genes, It is no doubt that would
increase the efficiency of subtractive hybridization method by simplifying experimental procedures and reducing experimental
time-consuming. In this study, we reported a modified method, called transcriptome subtractive hybridization (abbr. TSH). In order to
cover different types of RNA as much as possible and reduce the experimental procedure, TSH in the protocol neither uses restriction
endonuclease for adopter ligation, nor use oligo-d (T) for the reverse transcription reaction. Instead, tester RNA directly hybridizes
with driver single strand cDNA, then the hybrid of RNA/cDNA was digested by
Hae
Ⅲ
, residual targeting tester RNA was enriched
by reverse transcription PCR amplification. As a result, the experimental protocol was simplified to reduce experimental
time-consuming.
To validate use reliability of the TSH, TSH was employed to identify cell-specific expressing ESTs in both
E. coli
JM109 with and
without a recombinant plasmid. The results showed that the non-target RNA finally eliminated through direct hybridization and
digestion of RNA/cDNA hybrids. Six reference genes within the recombinant plasmid were detected as expected in the tester library
constructed from the JM109 RNA with the recombinant plasmid.
Obviously, the verified results showed that the TSH, a modified protocol, would be a reliable and effective method, which can be
applied to identify differential gene expression between the two cell types.
Keywords
Transcriptome subtractive hybridization; Differential expression; Reliability; An
E. coli
model
Background
Several methods are available to identify gene
differential expression between two types of different
cells, such as developed and undeveloped cells, or
treated and untreated cells. The approach called
suppression subtractive hybridization (SSH) is well
known used in the fields of Biosciences.
Duplex-specific nuclease (DSN), firstly isolated from
the
Kamchatka crab
(Veronika et al., 2008) can be able
to cleave double-stranded DNA or DNA in DNA-RNA
hybrids, but not to cleave single-stranded DNA or single
or double-stranded RNA. Due to specific functions of
the DSN, the SSH technique can enrich over 1 000
-
fold
DNAs from rare sequences in a single round of
subtractive hybridization (Rebrikov et al., 2004).
Recently, by using SSH technology, many target genes
were identified from specific cells in different species,
such as melanoma (Landreville et al., 2011), amaranth
(Aguilar-Hernández et al., 2011), beetle (Vogel et al.,
2011) and mycorrhiza (Murat et al., 2011). Generally,
there are 10 000~20 000 rare expressed mRNAs in a
typical cell accounting for only less than 20% of the
mass of mRNA (Carninci et al., 2000). Although
SSH works well in all of these reported studies, it is
not yet clear how deep the SSH could dig in the
transcriptome of a cell.
To reach more than 0.01% of the fractional concentra-
tion of a target gene in a cell by effective enrichment
in SSH, the concentration ratio (
R
) of a target gene
requires more than 5 folds between two cDNA prepa-