Affymetrix GeneChip Arabidopsis Genome [ATH1-121501]

Defining the Mitochondrial Stress Response in Arabidopsis thaliana

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Seven-day-old white-light-grown Arabidopsis seedlings were exposed for 15 minutes to polychromatic radiation with decreasing short-wave cut-off in the UV range, transferred back to the standard growth chamber and samples were taken 1 and 6 hours after the start of irradiation.

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UV-B quality

Transcription profiling of Arabidopsis response to UV-B

Seven-day-old white-light-grown Arabidopsis seedlings were exposed for 15 minutes to polychromatic radiation with decreasing short-wave cut-off in the UV range, transferred back to the standard growth chamber and samples were taken 1 and 6 hours after the start of irradiation.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-17438

Seedlings of Arabidopsis were irradiated for 15 minutes at midday with UV-B from Philips TL 40W/12 UV fluorescent tubes (Lambda,max = 310 nm, half-bandwidth = 40 nm, fluence rate = 7 W/m2) filtered through 3-mm transmission cut-off filters of the WG series with half-maximal transmission at the indicated wavelength (WG295, WG305, and WG327; Schott, Germany), or unfiltered through a 3-mm quartz plate. Spectral energy distributions of UV-B sources were measured with an OL 754 UV-visible spectroradiometer (Optronic Laboratories, Orlando, FL). After irradiation of the seedlings for the indicated times, plates were immediately returned to the standard growth chamber until the tissue was harvested and snap-frozen in liquid nitrogen.

P-MEXP-9939

Arabidopsis seeds were surface-sterilized with sodium-hypochlorite, plated on Murashige and Skoog medium (MS, Duchefa) containing 1% sucrose and 0.8% agar. Seeds were stratified for at least two days at 4°C and germinated aseptically at 25°C in a standard growth chamber (MLR-350, Sanyo Electric Co., Ltd.) with a 12 h/12 h light/dark cycle. 

P-MEXP-9941

Arabidopsis total RNA was isolated with the Plant RNeasy Kit (Qiagen, Chatsworth, CA), according to the manufacturer’s instructions.

P-MEXP-9943

Whole plant tissue of at least 50 Arabidopsis seedlings was harvested, snap-frozen in liquid nitrogen and stored at -80°C until further processing.

P-MEXP-9948

10 µg of total RNA (isolated from approx. 50 7-day-old Arabidopsis seedlings) was reverse transcribed using the SuperScript Choice system for cDNA synthesis (Life Technologies) according to the protocol recommended by Affymetrix (GeneChip Expression Analysis: Technical Manual (2001) p. 2.1.14-2.1.16). The oligonucleotide used for priming was 5’-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3’ (Genset Oligo, France) as recommended by Affymetrix. Double-stranded cDNA was purified by phenol:chloroform extraction and the aqueous phase was removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics, USA) following the manufacturer’s protocol. The cRNA was purified using RNAeasy clean-up columns (Qiagen). To improve the recovery from the columns, the elution water was spun into the matrix at 27 g and then left to stand for one minute prior to the standard 8000 g centrifugation recommended by Qiagen. The cRNA was fragmented by heating in fragmentation buffer (40mM Tris-acetate pH8.1, 100mM KOAc, 30mM MgOAc) as recommended by Affymetrix.

Transcription profiling of Arabidopsis response to UV-B

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