Arabidopsis Microarray Gene Expression Analysis

Microarrays were performed according to a previously described procedure [30 (link)]. Young buds collected from WT and 5mARF17/WT plants were immediately frozen in liquid nitrogen. A Low-RNA-Input Linear Amplification Kit (Agilent Technologies) was used to amplify and label the total RNA. 5-(3-Aminoallyl)-UTP (Ambion), Cy3 NHS ester (GE Healthcare Biosciences) and Cy5 NHS ester (GE Healthcare Biosciences) were applied following the manufacturers’ instructions. The labeled cRNA was purified using an RNeasy Mini Kit (Qiagen). According to the manufacturer’s instructions, each 44-K Arabidopsis oligo microarray slide was hybridized with 825 ng of Cy3-labeled cRNA and 825 ng of Cy5-labeled cRNA using a gene expression hybridization kit (Agilent) in a hybridization oven (Agilent). The slides were scanned using an Agilent Microarray Scanner (Agilent) and Feature Extraction software 10.7 (Agilent) with the default settings. Three biological replicates of independently grown materials were used. The raw data were normalized with a locally weighted scatter plot smoothing (Lowess) algorithm using Gene Spring Software 11.0 (Agilent).

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Wang B., Xue J.S., Yu Y.H., Liu S.Q., Zhang J.X., Yao X.Z., Liu Z.X., Xu X.F, & Yang Z.N. (2017). Fine regulation of ARF17 for anther development and pollen formation. BMC Plant Biology, 17, 243.

Publication 2017

Low-RNA-Input Linear Amplification Kit 5-(3-Aminoallyl)-UTP Cy3 NHS ester Cy5 NHS ester RNeasy Mini Kit gene expression hybridization kit hybridization oven Agilent Microarray Scanner Feature Extraction software 10.7 Gene Spring Software 11.0

Arabidopsis Biological Crna Ester Frozen Gene Gene expression Hybridization Microarray Nitrogen Oligo Plants

Corresponding Organization :

Other organizations : Tongji University, Shanghai Normal University

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Variable analysis

independent variables

Genotype (WT and 5mARF17/WT plants)

dependent variables

Gene expression (measured by microarray analysis)

control variables

Experimental procedure (previously described procedure [30])
RNA amplification and labeling (Low-RNA-Input Linear Amplification Kit, 5-(3-Aminoallyl)-UTP, Cy3 NHS ester, Cy5 NHS ester)
Microarray hybridization (gene expression hybridization kit, hybridization oven)
Microarray scanning (Agilent Microarray Scanner, Feature Extraction software 10.7)
Data normalization (Lowess algorithm, Gene Spring Software 11.0)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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