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Sample GSM1231696 Query DataSets for GSM1231696
Status Public on Nov 01, 2013
Title flight vs ground - rep 4
Sample type RNA
 
Channel 1
Source name Ground GAP 11, FPA 4
Organism Candida albicans
Characteristics strain: SC5413
growth condition: ground-based control condition
Growth protocol Prior to flight, 6 x 10^6 cells grown in YPD medium were suspended in 0.5 mL sterile ddH2O and loaded into specialized spaceflight hardware, termed Fluid Processing Apparatuses (FPA. Briefly, growth was initiated in flight (nine days post launch) by addition of 2 mL YPD to the fungal suspension (termed activation). Cultures were grown in spaceflight conditions or synchronous ground control conditions for 25 hours at ambient temperature (23˚C). Subsequently, cells were fixed for RNA, proteins and morphological imaging by addition of 2.5 mL RNA Later II reagent (Ambion, Austin, TX) (termed termination).)
Extracted molecule total RNA
Extraction protocol Two and a half hours after landing at Kennedy Space Center, the culture samples fixed in RNA Later II were recovered, removed from the FPA, and stored at -80°C. Yeast cells were disrupted by homogenization in the presence of glass beads in a Mini-Beadbeater-8™ (Biospec Products) and RNA was isolated using the RNeasy Micro kit (Qiagen). RNA quality and quantity were evaluated using the Nanodrop technology (Thermo Scientific) and an Agilent 2100 bioanalyzer (Agilent Technologies).
Label Cy3
Label protocol First strand cDNA is generated by oligo-dT primed reverse transcription (Superscript II; Invitrogen) utilizing the 3DNA Array 350 kit (Genisphere). Modified oligo-dT primers are utilized in which a fluorophore/dendrimer specific oligo sequence is attached to the 5’ end of the dT primer. For cDNA synthesis, 1ul of fluorophore specific oligo-dT primer is added to 8ug of total RNA and the solution is incubated at 80C for 10 minutes then cooled on ice for 2 minutes. To each sample are added RNase inhibitor (Superase-In; Ambion) (1ul), 5X first strand buffer (4ul), dNTP mix (10mM each dATP, dCTP, dGTP, and dTTP) (1ul), 0.1M DTT (2ul), and Superscript II RNase H- Reverse Transcriptase (1ul). Reverse transcription is carried out at 42C for 2 hours. The reaction is terminated by adding 0.5M NaOH/ 50mM EDTA (3.5ul) and incubation at 65C for 15 minutes then neutralized with 1M Tris-HCL, pH 7.5 (5ul). For RNA expression level comparison, samples are paired and concentrated using Microcon YM30 microconcentrators (Millipore) according to the manufacturer’s protocol.
 
Channel 2
Source name Flight GAP 11, FPA 5
Organism Candida albicans
Characteristics strain: SC5413
growth condition: spaceflight condition
Growth protocol Prior to flight, 6 x 10^6 cells grown in YPD medium were suspended in 0.5 mL sterile ddH2O and loaded into specialized spaceflight hardware, termed Fluid Processing Apparatuses (FPA. Briefly, growth was initiated in flight (nine days post launch) by addition of 2 mL YPD to the fungal suspension (termed activation). Cultures were grown in spaceflight conditions or synchronous ground control conditions for 25 hours at ambient temperature (23˚C). Subsequently, cells were fixed for RNA, proteins and morphological imaging by addition of 2.5 mL RNA Later II reagent (Ambion, Austin, TX) (termed termination).)
Extracted molecule total RNA
Extraction protocol Two and a half hours after landing at Kennedy Space Center, the culture samples fixed in RNA Later II were recovered, removed from the FPA, and stored at -80°C. Yeast cells were disrupted by homogenization in the presence of glass beads in a Mini-Beadbeater-8™ (Biospec Products) and RNA was isolated using the RNeasy Micro kit (Qiagen). RNA quality and quantity were evaluated using the Nanodrop technology (Thermo Scientific) and an Agilent 2100 bioanalyzer (Agilent Technologies).
Label Cy5
Label protocol First strand cDNA is generated by oligo-dT primed reverse transcription (Superscript II; Invitrogen) utilizing the 3DNA Array 350 kit (Genisphere). Modified oligo-dT primers are utilized in which a fluorophore/dendrimer specific oligo sequence is attached to the 5’ end of the dT primer. For cDNA synthesis, 1ul of fluorophore specific oligo-dT primer is added to 8ug of total RNA and the solution is incubated at 80C for 10 minutes then cooled on ice for 2 minutes. To each sample are added RNase inhibitor (Superase-In; Ambion) (1ul), 5X first strand buffer (4ul), dNTP mix (10mM each dATP, dCTP, dGTP, and dTTP) (1ul), 0.1M DTT (2ul), and Superscript II RNase H- Reverse Transcriptase (1ul). Reverse transcription is carried out at 42C for 2 hours. The reaction is terminated by adding 0.5M NaOH/ 50mM EDTA (3.5ul) and incubation at 65C for 15 minutes then neutralized with 1M Tris-HCL, pH 7.5 (5ul). For RNA expression level comparison, samples are paired and concentrated using Microcon YM30 microconcentrators (Millipore) according to the manufacturer’s protocol.
 
 
Hybridization protocol Each sample pair (~5ul) is resuspended in Formamide-based hybridization buffer (vial 7-Genisphere) (26ul), Array 50dT blocker (Genisphere) (2ul), and Rnase/Dnase-free water (19ul). Two hybridizations are carried out in a sequential manner. The primary hybridization is performed by adding 48ul of sample to the microarray under a supported glass coverslip (Erie Scientific) at 43C for 16-20 hours at high humidity. Prior to the secondary hybridization, slides are gently submerged into 2X SSC, 0.2% SDS (at 43C) for 11 min., transferred to 2X SSC (at room temp.) for 11 min., transferred to 0.2X SSC (at room temp.) for 11 min., and then spun dry by centrifugation. Secondary hybridization is carried out using the complimentary capture reagents provided in the 3DNA Array 350 kit (Genisphere). For each reaction, the following are added: 3DNA capture reagent with Cy3 (2.5ul), 3DNA capture reagent with Cy5 (2.5ul), SDS-based hybridization buffer (vial 6-Genisphere) (26ul), and Rnase/Dnase-free water (21ul). The secondary hybridization solution is incubated in the dark at 80C for 10 min. then 50C for 15 min. Hybridization is performed by adding 48ul secondary hybridization solution to the slide under a supported glass coverslip at 65C for 3 hr at high humidity in the dark. At hybridization termination, arrays are gently submerged into 2X SSC, 0.2% SDS (at 65C) for 11 min., transferred to 2X SSC (at room temp.) for 11 min., transferred to 0.2X SSC (at room temp.) for 11 min., and then spun dry by centrifugation. To prevent fluorophore degredation, the arrays are treated with Dyesaver (Genisphere).
Scan protocol Slides are scanned on a Perkin Elmer ScanArray Express HT scanner to detect Cy3 and Cy5 fluorescence. Laser power is kept constant for Cy3/Cy5 scans and PMT is varied for each experiment based on optimal signal intensity with lowest possible background fluorescence. A low pmt setting scan is also performed to recover signal from saturated elements. Gridding and analysis of images is performed using ScanArray v3.0 (Perkin Elmer).
Description slide 49
Data processing The median value of each set of replicate spots from each array was used. Data was log2 transformed and quantile normalized. The Ch2 N Log Ratio is the normalized log (base2) transformation of the ratio of the medians. These log ratios are cy5/cy3, or as listed in the raw data files, ch2/ch1. However, in the dye-swap samples, the values were inversed so that all sample data values represent flight/ground (test/reference) ratio. Three-way ANOVA analysis was then performed on the data using treatment (flight vs. ground), dye, and experimental data as factors. Flight to ground linear contrast was performed with ANOVA. False Discovery Rate was controlled using the Step Up method. The Candida Genome Database (CGD) gene ontology (GO) Slim Mapper was used to classify differentially expressed genes into functional categories.
Analysis was initially restricted to genes that had high intensity on the array and were differentially expressed by at least 2-fold with a confidence interval of 95%.
 
Submission date Sep 16, 2013
Last update date Nov 01, 2013
Contact name Sheila Nielsen
Phone (406) 994-1670
Organization name Montana State University
Department Immunology and Infectious Disease
Street address 317 Leon Johnson Hall
City Bozeman
State/province MT
ZIP/Postal code 59717
Country USA
 
Platform ID GPL9545
Series (1)
GSE50881 Candida albicans response to spaceflight (NASA STS-115)

Data table header descriptions
ID_REF
VALUE log2, quantile normalized ratio representing (flight/ground)

Data table
ID_REF VALUE
1 0.162
2 -1.837
3 1.185
4 -0.176
5 0.016
6 0.841
7 -1.111
8 0.009
9 -0.897
10 0.009
11 0.936
12 -0.164
13 -0.798
14 -0.942
15 1.374
16 -1.27
17 2.165
18 0.937
19 1.077
20 0.531

Total number of rows: 20160

Table truncated, full table size 232 Kbytes.




Supplementary file Size Download File type/resource
GSM1231696_Slide_49.csv.gz 2.9 Mb (ftp)(http) CSV
Processed data included within Sample table

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