tissue: green leaves genotype: Ye478, an inbred line treatment: 4mM nitrate age: leaves around 20 days after germination
Treatment protocol
Nitrate concentrations was 4 mM, which represented an optimal nitrate condition (high N). Ca (NO3)2.4H2O was used as the nitrate source. The other nutrients in solution were (in mmol L-1): 0.75 K2SO4, 0.1 KCl, 0.25 KH2PO4, 0.65 MgSO4.7H2O, and 0.2 EDTA-Fe, and in micromol L-1, 1.0 MnSO4.H2O, 1.0 ZnSO4.7H2O, 0.1 CuSO4.5H2O, and 0.005 (NH4)6Mo7O24.4H2O. Air was continuously pumped through the solution (pH 6.0) that was changed every 2 days. Seedlings were sampled after 15 days for RNA extraction.
Growth protocol
Seeds of Ye478 were sterilized with 10% (v/v) H2O2 for 30 min, washed with distilled water, soaked in saturated CaSO4 for 6 h, and then germinated at 28 degree centigrade for 2 d in the dark between two layers of filter paper moistened with saturated CaSO4. Seeds with 1-2 cm germ were transferred to coarse silica sand to grow at 28 degree centigrade /22 degree centigrade during the 14/10 h light / dark cycle. Uniform seedlings with two visible leaves were selected. The residual endosperms of the seedlings were discarded. Plants were planted in a glass beaker. The outside of the glass beakers were covered with a black sheet to ensure that the roots were kept in complete darkness. Glass beakers containing ten seedlings were maintained in an illumination chamber.
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
Cy3
Label protocol
Sample was labled with CU-Cy3 (Dharmacon, USA) using T4 ligase according to the method of the artical (Thomson,J.M., Parker,J., Perou,C.M., Hammond,S.M. (2004). A custom microarray platform for analysis of microRNA gene expression. Nat.Methods 1, 47-53.). Then, labled sample was precipitated with 100% ethyl alcohol and blew dry.
tissue: green leaves genotype: Ye478, an inbred line treatment: 0.04mM nitrate age: leaves around 20 days after germination
Treatment protocol
Nitrate concentrations was 0.04 mM, which represented low-nitrate availability (low N) with Ca (NO3)2.4H2O used as the nitrate source. Ca was compensated to 2 mM at low N with CaCl2 . The other nutrients in solution were (in mmol L-1): 0.75 K2SO4, 0.1 KCl, 0.25 KH2PO4, 0.65 MgSO4.7H2O, and 0.2 EDTA-Fe, and in micromol L-1, 1.0 MnSO4.H2O, 1.0 ZnSO4.7H2O, 0.1 CuSO4.5H2O, and 0.005 (NH4)6Mo7O24.4H2O. Air was continuously pumped through the solution (pH 6.0) that was changed every 2 days. Seedlings were sampled after 15 days for RNA extraction.
Growth protocol
Seeds of Ye478 were sterilized with 10% (v/v) H2O2 for 30 min, washed with distilled water, soaked in saturated CaSO4 for 6 h, and then germinated at 28 degree centigrade for 2 d in the dark between two layers of filter paper moistened with saturated CaSO4. Seeds with 1-2 cm germ were transferred to coarse silica sand to grow at 28 degree centigrade /22 degree centigrade during the 14/10 h light / dark cycle. Uniform seedlings with two visible leaves were selected. The residual endosperms of the seedlings were discarded. Plants were planted in a glass beaker. The outside of the glass beakers were covered with a black sheet to ensure that the roots were kept in complete darkness. Glass beakers containing ten seedlings were maintained in an illumination chamber.
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
Cy5
Label protocol
Sample was labled with CU-Cy5 (Dharmacon, USA) using T4 ligase according to the method of the artical (Thomson,J.M., Parker,J., Perou,C.M., Hammond,S.M. (2004). A custom microarray platform for analysis of microRNA gene expression. Nat.Methods 1, 47-53.). Then, labled sample was precipitated with 100% ethyl alcohol and blew dry.
Hybridization protocol
For microarray hybridization, each probe is printed in triplicate using a SmartArray microarrayer (CapitalBio). Labled RNA was resuspended in 16 microliter hybridization solution containing 15% formamide, 0.2% SDS, 3 fold SSC and 50 fold Denhardt's. The hybridization was performed at 42 degree centigrade for overnight, and then washed in a solution containing 2 fold SSC and 0.2% SDS at 42 degree centigrade for 4 min. And then washed with 0.2 fold SSC solution at room temperature for 4 min and spin-drying.
Scan protocol
Slides were scanned with LuxScan 10K/A scanner (CapitalBio) and raw pixel intensities were extracted with Lux- Scan 3.0 software.
Data processing
In order to make every slides have the same global mean, linear calibration was performed between different slides according to the global mean of Cy5 and Cy3 signal. After that, linear nomarlization within silde was performed using Lowess way accroding to the artical (Yang,Y.H.,et al., space and intensity-dependant normalization, Nucleic Acids Research, 2002,30:e15). The ratio between two samples was calculated with the normalized signal of Cy3 and Cy5, and then the log value based with 10 of the ratio was calculated as the final VALUE in the data table.
The levels of significance of differentially expressed miRNAs were analyzed using Significance Analysis of Microarrays software (SAM, version 3.02, http://www-stat.stanford.edu/~tibs/SAM/) (Stanford University, USA). MiRNAs with q<0.01 and Ratio >2 or <0.5 were defined as differentially expressed miRNAs.
