Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
Identifieur interne : 000245 ( Main/Exploration ); précédent : 000244; suivant : 000246Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
Auteurs : Anna Karlgren [Suède] ; Jenny Carlsson ; Niclas Gyllenstrand ; Ulf Lagercrantz [Suède] ; Jens F. SundströmSource :
- Journal of Visualized Experiments : JoVE [ 1940-087X ] ; 2009.
Abstract
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se
Url:
DOI: 10.3791/1205
PubMed: 19377440
PubMed Central: 3148633
Affiliations:
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Le document en format XML
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Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species</title>
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Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species</title>
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<front><div type="abstract" xml:lang="en"><p>The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. <italic>in situ</italic>
hybridization, a technique used to localize cell specific mRNA expression. The <italic>in situ</italic>
hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. <italic>In situ</italic>
experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (<italic>Picea abies</italic>
). Here we present a modified DIG <italic>in situ</italic>
hybridization protocol, which is fast and applicable on a wide range of plant species including <italic>P. abies</italic>
. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; <italic>P. abies, Arabidopsis thaliana</italic>
and <italic>Brassica napus</italic>
. The protocol worked equally well for the species and genes studied. <italic>AtAP3</italic>
and <italic>BnAP3</italic>
were observed in second and third whorl floral organs in <italic>A. thaliana</italic>
and <italic>B. napus</italic>
and DAL13 in microsporophylls of male cones from <italic>P. abies</italic>
. For <italic>P. abies</italic>
the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG <italic>in situ</italic>
protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.</p>
<p>Anna Karlgren and Jenny Carlsson contributed equally to this study.</p>
<p>Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se</p>
</div>
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