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Sample GSM367014 Query DataSets for GSM367014
Status Public on Dec 27, 2012
Title Bme-inf HeLa_4h_rep1_HeLa array
Sample type RNA
 
Channel 1
Source name Bmel-infected HeLa cells, total RNA, 4h p.i., Cy5
Organism Homo sapiens
Characteristics Cell line: S3 (ATCC; CCL 2.2)
Extracted molecule total RNA
Extraction protocol Total RNA from infected and non-infected HeLa cell cultures was extracted by TRI-Reagent® (Ambion, Austin, TX) according to manufacturer’s instructions. The resultant RNA pellet was re-suspended in DEPC-treated water with 2% DTT and 1% RNase inhibitor. Contaminant genomic DNA was removed by RNase-free DNase I treatment according to the manufacturer’s instructions, and samples were stored at -80ºC until used.
Label Cy5
Label protocol Ten μg of total RNA were reverse transcribed overnight to amino-allyl cDNA using 6 µg of random hexamer primers, 0.6 μl 50X dNTPs/ aa-dUTP mix (2:3 aa-dUTP:dTTP) and 400U Superscript III. The reaction was stopped by incubating the samples with 1M NaOH at 65ºC for 15 min and neutralized with 1M HCl. Unincorporated aa-dUTPs and free amines were removed by column passage and dried using a Speed-Vac. Dried samples were re-hydrated in 0.1M Na2CO3 buffer (pH 9.0) and labeled with Cy5-ester (experimental HeLa RNA) or Cy3-ester (reference HeLa RNA). After one hour incubation in the dark, uncoupled dye was removed using PCR purification kit and dye incorporation calculated by NanoDrop® ND-1000 (NanoDrop). The dried, labeled cDNA samples were re-suspended in 20 μl of nuclease-free water (experimental RNA) and human genomic DNA Cot1 (reference RNA), mixed and heated at 95ºC for 10 min, 60ºC for 10 min, and then 25ºC for 10 min. Samples were kept at 45ºC until hybridization. Immediately before hybridization, 40 μl of 2X formamide-based hybridization buffer was added to each sample.
 
Channel 2
Source name Universal Human Reference RNA labeled with Cy3 (green).
Organism Homo sapiens
Characteristics Several human cell lines
Comercial source (Stratagene)
Extracted molecule total RNA
Extraction protocol Total RNA from infected and non-infected HeLa cell cultures was extracted by TRI-Reagent® (Ambion, Austin, TX) according to manufacturer’s instructions. The resultant RNA pellet was re-suspended in DEPC-treated water with 2% DTT and 1% RNase inhibitor. Contaminant genomic DNA was removed by RNase-free DNase I treatment according to the manufacturer’s instructions, and samples were stored at -80ºC until used.
Label Cy3
Label protocol Ten μg of total RNA were reverse transcribed overnight to amino-allyl cDNA using 6 µg of random hexamer primers, 0.6 μl 50X dNTPs/ aa-dUTP mix (2:3 aa-dUTP:dTTP) and 400U Superscript III. The reaction was stopped by incubating the samples with 1M NaOH at 65ºC for 15 min and neutralized with 1M HCl. Unincorporated aa-dUTPs and free amines were removed by column passage and dried using a Speed-Vac. Dried samples were re-hydrated in 0.1M Na2CO3 buffer (pH 9.0) and labeled with Cy5-ester (experimental HeLa RNA) or Cy3-ester (reference HeLa RNA). After one hour incubation in the dark, uncoupled dye was removed using PCR purification kit and dye incorporation calculated by NanoDrop® ND-1000 (NanoDrop). The dried, labeled cDNA samples were re-suspended in 20 μl of nuclease-free water (experimental RNA) and human genomic DNA Cot1 (reference RNA), mixed and heated at 95ºC for 10 min, 60ºC for 10 min, and then 25ºC for 10 min. Samples were kept at 45ºC until hybridization. Immediately before hybridization, 40 μl of 2X formamide-based hybridization buffer was added to each sample.
 
 
Hybridization protocol Slides were hybridized at 45ºC for ~20 h in a dark, humid chamber (Corning) and washed for 10 min at 45ºC with low stringency buffer [1X SSC, 0.2% SDS] followed by two 5-min washes in a higher stringency buffer [0.1X SSC, 0.2% SDS and 0.1X SSC] at room temperature with agitation. Slides were dried by centrifugation at 800X g for 2 min and immediately scanned. Prior to hybridization, microarrays were pre-treated by washing in 0.2% SDS, followed by 3 washes in distilled water and incubated in prehybridization buffer [5X SSC, 0.1% SDS; 1% BSA in 100ml of water] at 45ºC for at least 45 min. Immediately before hybridization, the slides were washed 4 times in distilled water, dipped in 100% isopropanol for 10 sec and dried by centrifugation at 1,000X g for 2 min.
Scan protocol Microarrays were scanned using a commercial laser scanner (GenePix 4100; Axon Instruments Inc., Foster City, CA). The genes represented on the arrays were adjusted for background and normalized to internal controls using image analysis software (GenePixPro 6.0; Axon Instruments Inc.).
Description Biological replicate 1 of 4. Total RNA from BME-infected HeLa cells at 4h p.i. co-hybridized against universal human reference RNA
Data processing Genes with fluorescent signal values below background were disregarded in all analyses. Host arrays were initially normalized against universal human reference RNA (Weil et al., 2002) and resulting data analyzed using Seralogix’s suite of gene expression analysis and modeling tools (www.seralogix.com). Differentially expressed genes were found to be significant based on Seralogix’s Bayesian z-score method (z > 1.96 or equivalently p < 0.05). This method infers a given gene’s standard deviation by computing a local average standard deviation for genes showing similar expression levels. Genes are ordered within a treatment group according to their average expression levels. Then, for a given gene, the k next higher expressing genes and the k next lower expressing genes standard deviations are used to obtain an average standard deviation of these 2k + 1 genes. This approach results in a nonparametric smoothing function for inferring any given gene’s standard deviation from its expression level. For this study k = 50 was utilized. The inferred standard deviations are derived for each time point and condition (infected and controls) and used in a z-score method to identify significant differentially expressed genes.
 
Submission date Feb 04, 2009
Last update date Dec 27, 2012
Contact name Leslie Garry Adams
E-mail(s) gadams@cvm.tamu.edu
Phone 979-845-9816
Organization name Texas A&M University
Department Veterinary Pathobiology
Lab L. Garry Adams Lab
Street address 402 Raymond Stotzer Blvd.
City College Station
State/province TX
ZIP/Postal code 77843-4467
Country USA
 
Platform ID GPL8155
Series (1)
GSE14703 Brucella melitensis-infected HeLa cells gene expression

Data table header descriptions
ID_REF
VALUE Normalized log2 ratio (Cy5/Cy3) via Weil MR et al 2002

Data table
ID_REF VALUE
1 0.073914037
2 0.458043585
3 -0.470667847
4 0.359685696
5 -0.33795882
6 0.229820898
7 0.185588101
8 0.151626067
9 0.404127164
10 0.220219794
11 0.377854357
12 0.585347938
13 -0.042414746
14 0.064177446
15 -0.628116674
16 0.036471749
17 -0.479441778
18 1.113937621
19 -0.002216714
20 0.192116881

Total number of rows: 9767

Table truncated, full table size 163 Kbytes.




Supplementary file Size Download File type/resource
GSM367014.gpr.gz 1.1 Mb (ftp)(http) GPR
Processed data included within Sample table

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