15 protocols
Title: Affymetrix CEL analysis. Description:
hybridization protocol
Affymetrix Generic Hybridization
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
Lymphocytes isolated from two healthy donors (D1 - donor 1 and D2 - donor 2) were used for gene expression analysis following low and high dose radiation. Lymphocytes were isolated from a 50 ml buffy-coat using Ficoll-Paque plus according to manufacturer’s protocol (Amersham Biosciences, Sweden). Isolated lymphocytes were stored in cryo-tubes (50-106 cells in each) at -140C in lymphocyte medium as described below, containing 10% DMSO. Before radiation cells were thawed and incubated in a 75 cm2 flask overnight in 15 ml complete medium (RPMI 1640 (Dutch modification) with 25 mM HEPES, supplemented with 20% AIM-V serum free medium (Gibco), 15% heat-inactivated defined bovine calf serum (Hyclone), 10% LAK supernatant[16] , 50 ?M ß-mercaptoethanol, 2 mM L-glutamax, (Gibco), 1 mM Na-pyruvate (Gibco) and antibiotics (final concentration 100 units/ml penicillin, 100 ?g/ml streptomycin sulphate).

Non-adherent cells were collected on the following day. Cells were counted and diluted to a concentration of 5×106/ml after which they were stimulated to grow by addition of PHA-HA16 (Murex, Dartford, UK) at a final concentration of 1 µg/ml followed by 48h incubation at 37 C. Cells were counted and suspended into 0.25×106/ml with a reduced concentration of PHA-HA16 of 0.5 µg/ml. The cell suspension was subdivided over 10 cell culture flasks containing 8 ml each (25 cm2) and irradiated with ?-rays from a 137Cs source at a dose rate of 0.56 Gy/min (Scanditronix, Uppsala, Sweden) with doses of 25 mGy, 50 mGy, 100 mGy, 200 mGy and 2 Gy (Fig. 1 in Supplementary Material). Control samples were mock irradiated. Following irradiation all samples were incubated for 24 h at 37 C after which total RNA was isolated using the standard protocol provided by Qiagen.

Radiation of lymphocytes and RNA isolation was performed separately for each donor in three independent irradiation experiments per donor and are referred to in further analysis as experimental repeats (D1a-c, D2 d-f).

(Parameters: start time = 72, time unit = hours, min temperature = 37, temperature unit = C, media = Lymphocyte medium)
Qiagen total RNA isolation protocol
(Parameters: Extracted product = total_RNA, Amplification = none)
RMA normalized data. A custom Chip Definition Files (CDF) developed by Ferrari et al (Ferrari F, Bortoluzzi S, Coppe A, Sirota A, Safran M, Shmoish M et al.: Novel definition files for human GeneChips based on GeneAnnot. BMC Bioinformatics) was used for gene annotation.
A Cy5-labeled reference library was generated by 10 cycles of PCR using 5' Cy5-17.93D (/Cy5/ATCGTAGGCACCTGAAA) and 17.92_c18_A20 (AAAAAAAAAAAAAAAAAAAA/iSp18/ATTGATGGTGCCTACAG) using a 45nM pool containing equal amounts of all 225 reference oligonucleotides as template. [Reference oligos are complimentary to all spots on the At_Dm_Ce_1k_v1_g1_2 array and contain constant 5' and 3' flanking adaptors to mimic small RNA cDNAs; See Axtell and Bartel for details] Labeled PCR products were fractionated through a 6% denaturing polyacrylamide gel, enabling excision of the shorter Cy5-labeled strands. Samples were adjusted to 5 µM in water.
Small RNAs from 50 ug of a total RNA sample were fractionated, sequentially ligated to 3' and 5' adapters, and reverse transcribed as described by (Lau et al. 2001. Science 294:858-862). First-stage PCR used oligos 17.92 and 17.93D (Lau et al. 2001. Science 294:858-862), and proceeded until amplifications were in linear stage (as determined by visualization of products from successive cycles); typically 17-19 cycles. A 1/100 dilution of this reaction was used as template in a labeling PCR using oligos 5' Cy3-17.93D (/5Cy3/ATCGTAGGCACCTGAAA) and 17.92_c18_A20 (AAAAAAAAAAAAAAAAAAAA/iSp18/ATTGATGGTGCCTACAG) for 10 cycles to create an asymmetric PCR product. Labeled PCR products were fractionated through a 6% denaturing polyacrylamide gel, enabling excision of the shorter Cy3-labeled strands. Samples were adjusted to 5 µM in water.
2 µl of 5 µM Cy3-labeled sample and 2 µl of 5 µM Cy5-labeled reference was added to 20 µl of hybridization buffer (3.5X SSC, 1% [m/v] BSA, 0.1% [m/v] SDS, 93 µg/ml salmon testes DNA, 187 µg/ml Escherichia coli tRNA, 37 µg/ml poly-adenine) for a final concentration of 0.417 µM each. After heating for 4 minutes at 85°C, samples were applied to arrays that had been pre-hybridized for 45 minutes in 3.5X SSC, 1% (m/v) BSA, 0.1% (m/v) SDS, rinsed with de-ionized water, and dried. Arrays were incubated at 57°C for 16 hrs, then washed for 5 minutes at 50°C in 2X SSC, 0.1% SDS, followed by 10 minutes at room temperature in 0.1X SSC, 0.1% SDS, and 3 x 1 minute at room temperature in 0.1X SSC.
10 µm per pixel, line average 2, and constant PMT gains for both 635 nm and 532 nm.
Described in Mallory et al., Plant Cell 13:571-583 (2001).
Long-day conditions at 18C. 16 hours light, 8 hours dark. Organs harvested when 50-60 days old.
Growth at 18C in 8 hr light, 16 hr dark. Seedlings harvested 10 days after sowing.
Growth at 18C for 16hr light, 8 hr dark. Seedlings harvested after 10 days.
Arabidopsis root RNA samples were derived from Col-0 roots harvested from 14 day old plants grown in constant light in liquid culture (1X Murashige {MS} salts + vitamins, 1% sucrose, 5 mM MES-KOH pH 5.7) shaking at 60 rpm in constant light at 22°C.