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Profiling Gene Expression in Germinating Brassica Roots.

Park MR, Wang YH, Hasenstein KH - Plant Mol. Biol. Rep. (2014)

Bottom Line: The expression level of all genes differed significantly at each sample position.SPGE needles can be used within two weeks when stored at 4 °C.Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

View Article: PubMed Central - PubMed

ABSTRACT
Based on previously developed solid-phase gene extraction (SPGE) we examined the mRNA profile in primary roots of Brassica rapa seedlings for highly expressed genes like ACT7 (actin7), TUB (tubulin1), UBQ (ubiquitin), and low expressed GLK (glucokinase) during the first day post-germination. The assessment was based on the mRNA load of the SPGE probe of about 2.1 ng. The number of copies of the investigated genes changed spatially along the length of primary roots. The expression level of all genes differed significantly at each sample position. Among the examined genes ACT7 expression was most even along the root. UBQ was highest at the tip and root-shoot junction (RS). TUB and GLK showed a basipetal gradient. The temporal expression of UBQ was highest in the MZ 9 h after primary root emergence and higher than at any other sample position. Expressions of GLK in EZ and RS increased gradually over time. SPGE extraction is the result of oligo-dT and oligo-dA hybridization and the results illustrate that SPGE can be used for gene expression profiling at high spatial and temporal resolution. SPGE needles can be used within two weeks when stored at 4 °C. Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

No MeSH data available.


Related in: MedlinePlus

LabChip® 90 electropherograms of DEPC-treated water (a), total RNA extracted from roots of Brasscia seedlings (b), messenger RNA (mRNA) extracted from total RNA (c), and mRNA extracted from total RNA by SPGE (d). Peaks represent the lead marker (50 nt, arrow head) or ribosomal RNAs
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Fig1: LabChip® 90 electropherograms of DEPC-treated water (a), total RNA extracted from roots of Brasscia seedlings (b), messenger RNA (mRNA) extracted from total RNA (c), and mRNA extracted from total RNA by SPGE (d). Peaks represent the lead marker (50 nt, arrow head) or ribosomal RNAs

Mentions: The absorbance ratio (260/280 nm) of total RNA extracted directly from roots of Brassica and mRNAs isolated from total root RNA (2.5 μg/μl RNA) were 2.0 and 2.1, respectively. Labchip electropherograms of DEPC-treated water showed only a marker peak (Fig. 1a). The quantification of 28S and 18S (rRNA) on agarose gels and Labchip electropherograms showed that the total RNA was of good quality (Fig. 1b). mRNA comprised 0.97 % of total RNA. Primary rRNA (5 s, 18 s, and 28 s) peaks were not found on electropherogram of mRNA (Fig. 1c) or of mRNA extracted by SPGE (Fig. 1d). The largest fragments from total RNA, bulk-purified mRNA, and SPGE-extracted mRNA were 7,100, 6,200, and 4,400 nt, respectively (Fig. 1b, c, and d).Fig. 1


Profiling Gene Expression in Germinating Brassica Roots.

Park MR, Wang YH, Hasenstein KH - Plant Mol. Biol. Rep. (2014)

LabChip® 90 electropherograms of DEPC-treated water (a), total RNA extracted from roots of Brasscia seedlings (b), messenger RNA (mRNA) extracted from total RNA (c), and mRNA extracted from total RNA by SPGE (d). Peaks represent the lead marker (50 nt, arrow head) or ribosomal RNAs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3926982&req=5

Fig1: LabChip® 90 electropherograms of DEPC-treated water (a), total RNA extracted from roots of Brasscia seedlings (b), messenger RNA (mRNA) extracted from total RNA (c), and mRNA extracted from total RNA by SPGE (d). Peaks represent the lead marker (50 nt, arrow head) or ribosomal RNAs
Mentions: The absorbance ratio (260/280 nm) of total RNA extracted directly from roots of Brassica and mRNAs isolated from total root RNA (2.5 μg/μl RNA) were 2.0 and 2.1, respectively. Labchip electropherograms of DEPC-treated water showed only a marker peak (Fig. 1a). The quantification of 28S and 18S (rRNA) on agarose gels and Labchip electropherograms showed that the total RNA was of good quality (Fig. 1b). mRNA comprised 0.97 % of total RNA. Primary rRNA (5 s, 18 s, and 28 s) peaks were not found on electropherogram of mRNA (Fig. 1c) or of mRNA extracted by SPGE (Fig. 1d). The largest fragments from total RNA, bulk-purified mRNA, and SPGE-extracted mRNA were 7,100, 6,200, and 4,400 nt, respectively (Fig. 1b, c, and d).Fig. 1

Bottom Line: The expression level of all genes differed significantly at each sample position.SPGE needles can be used within two weeks when stored at 4 °C.Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

View Article: PubMed Central - PubMed

ABSTRACT
Based on previously developed solid-phase gene extraction (SPGE) we examined the mRNA profile in primary roots of Brassica rapa seedlings for highly expressed genes like ACT7 (actin7), TUB (tubulin1), UBQ (ubiquitin), and low expressed GLK (glucokinase) during the first day post-germination. The assessment was based on the mRNA load of the SPGE probe of about 2.1 ng. The number of copies of the investigated genes changed spatially along the length of primary roots. The expression level of all genes differed significantly at each sample position. Among the examined genes ACT7 expression was most even along the root. UBQ was highest at the tip and root-shoot junction (RS). TUB and GLK showed a basipetal gradient. The temporal expression of UBQ was highest in the MZ 9 h after primary root emergence and higher than at any other sample position. Expressions of GLK in EZ and RS increased gradually over time. SPGE extraction is the result of oligo-dT and oligo-dA hybridization and the results illustrate that SPGE can be used for gene expression profiling at high spatial and temporal resolution. SPGE needles can be used within two weeks when stored at 4 °C. Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

No MeSH data available.


Related in: MedlinePlus