|
| Status |
Public on May 24, 2018 |
| Title |
Late bicellular pollen, RNA from RNP complexes - replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Late bicellular pollen, RNA from RNP complexes
|
| Organism |
Nicotiana tabacum |
| Characteristics |
tissue: late bicellular pollen
cultivar: Samsun
|
| Treatment protocol |
No treatment
|
| Growth protocol |
Wild-type Nicotiana tabacum (cv. Samsun) seeds were sown in a greenhouse under 22-25°C and short-day conditions. Adult plants with fully developed roots were transplanted to an outdoor greenhouse to compost soil. There the plants were grown under the natural day-night photoperiod in spring and summer. Immature pollen grains at three stages of development were collected from August to September. The appropriate developmental stage was determined by the total length of flower buds including calyx as published previously by (Tupý J, Süss J, Hrabětová E and Říhová L: Biol Plant 25: 231-237, 1983). Flower bud lengths were as follows: uninuclear microspores (stage 1), 15-16 mm; early bicellular pollen (stage 3), 25-27 mm, late bicellular pollen (stage 5), 46-49 mm. After flower collection, the freshly isolated anthers were immediately processed by gentle mashing in a chilled mortar with 5% sucrose in sterile water. The mixture containing released pollen was filtered through an ordinary gauze to remove the anther debris. The pollen was then sedimented by centrifugation (2,000 g, 5 min, 4°C) and stored in -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total and subcellular fraction RNA was isolated from 100 mg of pollen using the Qiagen RNeasy Plant Kit according to the manufacturer’s instructions and treated with DNaseI (Promega). RNA was quantified using NanoDrop (Thermo Scientific). RNA concentration, purity and integrity (RIN) were assessed using an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3)-labeled cRNA was prepared from 66 ng of total RNA by Low Input Quick Amp Labeling Kit, One-Color (Agilent) according to the manufacturer's instruction. Cy3-labeled cRNA was purified by RNeasy Mini Kit (Qiagen). NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific) was used to evaluate the cRNA yield and labeling efficiency. Cy3-labeled cRNA was then fragmented in a 30-minute incubation at 60 °C.
|
| |
|
| Hybridization protocol |
1650 ng of Cy3-labeled cRNA was hybridized to Agilent Tobacco Gene Expression Microarray 4x44K for 17 h at 65 °C in a rotating Agilent hybridization oven (Agilent).
|
| Scan protocol |
The slides were scanned on the Agilent High Resolution Microarray Scanner using default parameters for 4x44K microarray formats (Agilent). Background-subtracted and spatially-detrended Processed Signal intensities were obtained by Feature Extraction Software 11.0.1.1 (Agilent) used with default parameters (Protocol GE1_1100_Jul11 and Grid 21113_D_F_20130122).
|
| Description |
Fraction: RNA RNA from RNP complexes
Gene expression profile of microspores
|
| Data processing |
All transcriptomic data sets were normalized using publicly available dChip 1.3 software (http://www.dchip.org). The reliability and reproducibility of the analyses were ensured by the use of duplicates in each experiment, the normalization of all arrays to the median probe intensity level, and the use of normalized intensities of all arrays for the calculation of model-based gene expression values based on the Perfect Match-only model (Li C and Wong WH: Proc Natl Acad Sci U S A 98: 31-36, 2001).
|
| |
|
| Submission date |
May 23, 2018 |
| Last update date |
May 24, 2018 |
| Contact name |
David Honys |
| E-mail(s) |
david.honys@gmail.com
|
| Phone |
420776352433
|
| Organization name |
IEB ASCR
|
| Lab |
Pollen Biology
|
| Street address |
Rozvojova 263
|
| City |
Prague 6 |
| State/province |
CZ |
| ZIP/Postal code |
16500 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL10098 |
| Series (1) |
| GSE114806 |
Tobacco pollen sequestrome dynamics |
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