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Series GSE2077 Query DataSets for GSE2077
Status Public on Feb 15, 2005
Title Gene expression in Cryptosporidium parvum-infected human ileocecal adenocarcinoma cells (HCT-8)
Organism Homo sapiens
Experiment type Expression profiling by array
Summary • The origin of biological samples (In vitro infection of HCT-8 cells with Cryptosporidium parvum)

Monolayers of the HCT-8 cells (ATCC CCL-244, American Type Culture Collection) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and additional nutritional supplements. Cells in log phase were plated at 2×106 cells per 150 mm tissue culture dish and infected with sterilised Cryptosporidium parvum oocysts (Iowa strain). Purified oocysts were suspended in phosphate buffered saline, sterilised in 33% bleach for 7 min on ice, washed in Hank's buffered saline solution, and added to HCT-8 cultures at a ratio of one oocyst per cell. Following a 2 h excystation period at 37 °C, cells were washed with warm Hank's buffered saline solution, and cells were incubated in fresh supplemented media. Mock-infected cultures were treated identically, with the exception that oocysts were not added to the cultures.

Three independent mock- and C. parvum-infected cultures were prepared for analysis.


• The origin of mRNA samples

At 6, 12, 24, 48, and 72h post-inoculation, cell culture media were removed and cultures were immediately lysed by the addition of 3.5 ml of Trizol® reagent (GIBCO BRL Life Technologies) directly to the culture plate. Total RNA was prepared as described by the manufacturer, and poly(A) mRNA was isolated using oligo-dT cellulose columns (Amersham Pharmacia Biotech). The qualities of total RNA and poly(A) mRNA preparations were assessed by Northern blot.


• Protocols for conducting microarray hybridization

Poly(A) mRNA was converted into ‘target’ suitable for hybridisation to Affymetrix microarray chips according to protocols provided by Affymetrix Inc. (Santa Clara, CA). Briefly, 2.0 g of poly(A) mRNA was reverse-transcribed to prepare double-stranded cDNA using T7-(dT)24 primer (GENSET Corp., La Jolla, CA) and Superscript II reverse transcriptase (GIBCO). Approximately 1 g of cDNA was used for in vitro transcription in the presence of biotinylated UTP and CTP using the Enzo BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Inc). This labelled cRNA was fragmented in 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate at 94 °C for 35 min. The integrity of cRNA and the efficiency of fragmentation were monitored by Northern blot analysis (not shown).
Fragmented biotinylated cRNA targets were hybridised to HG-U95Av2 chips (Affymetrix) that contain probe sets for 12,600 human gene/transcripts. Fifteen micrograms of fragmented cRNA and appropriate controls were hybridised to the chips at 45 °C for 16 h with constant rotation at 60 rev./min. The chips were subsequently washed and stained with streptavidin–phycoerythrin conjugate using the GeneChip Fluidics station protocol EukGE_WS2 (Affymetrix). Following washing and staining, microarray chips were scanned twice at 3 m resolution using a Hewlett-Packard confocal scanner and hybridisation intensities for each of the genes/transcripts were collected from scanned images.


• Analysis of microarray data

Gene expression data were initially analysed with the GeneChip® expression analysis software (Affymetrix Microarray Suite, version 5.0). The fluorescence intensity of the genes/transcripts was measured for each probe array and, to minimise discrepancies due to non-biological variations, normalised by global scaling to 1000.

Further data analyses were performed using GeneSpring software (version 6.0; Silicon Genetics, Redwood City, CA). Per gene normalisation was applied in which the expression signal of each gene in C. parvum-infected cells (raw data) was normalised to the median of its measurements in the mock-infected samples (control); and the ratio of expression levels between mock and infected samples was calculated as the mean value of normalised signal vs. control signal among three replicates. To identify genes with significantly altered expression, a series of statistical analyses (filtering) were performed: cut-off values for ratio of expression levels 1.80 and 0.55 were used to filter genes with expression level fold changes greater than ±1.8 in all three independent samples. Genes with fold change variations >1.5 across the three samples were excluded. Furthermore, a ‘statistical group comparison’ using Student's t-test/ANOVA was conducted to compare mean expression levels between mock-infected and infected samples, and the genes with significant differential expression (P<0.05) were selected. Identified differentially expressed genes were then annotated using GeneSpring's ‘Build Simplified Ontology’ constructor which hierarchically groups genes into meaningful biological categories (gene lists) based on the Gene Ontology Consortium Classifications. Various lists of regulated genes were created by cross-referencing annotated gene lists and applying assorted statistical and visualisation methods.
Keywords: time-course
 
 
Contributor(s) Deng M, Abrahamsen MS
Citation(s) 14711592
Submission date Dec 15, 2004
Last update date Dec 13, 2018
Contact name Mingqi Deng
E-mail(s) dengx010@umn.edu
Phone 612-625-7737
Fax 612-625-0204
Organization name University of Minnesota
Department Veterinary and Biomedical Sciences
Lab Mitchell Abrahamsen
Street address 1988 Fitch Avenue
City Saint Paul
State/province MN
ZIP/Postal code 55108
Country USA
 
Platforms (1)
GPL8300 [HG_U95Av2] Affymetrix Human Genome U95 Version 2 Array
Samples (18)
GSM37929 Cryptosporidium parvum-infected HCT-8 cells, time course 1, mock
GSM37930 Cryptosporidium parvum-infected HCT-8 cells, time course 1, 6h
GSM37931 Cryptosporidium parvum-infected HCT-8 cells, time course 1, 12h
Relations
BioProject PRJNA91281

Table 1: Cryptosporidium parvum-infected HCT-8 cells, mock (average of 3 replicates) header descriptions
ID_REF
Ratio
StdErr Norm
StdDev Norm
t-test P-value
Call
VALUE
StdErr Raw
StdDev Raw
Control
StdErr Control
StdDev Control

Data table
ID_REF Ratio StdErr Norm StdDev Norm t-test P-value Call VALUE StdErr Raw StdDev Raw Control StdErr Control StdDev Control
1000_at 1 0.11561946 0.1635106 1 P 4064.5 469.9353 664.58887 4064.5 0 0
1001_at 1 0.31710446 0.44845343 1 A 78.05 24.750002 35.00179 78.05 0 0
1002_f_at 1 0.14603949 0.20653102 1 A 153.75 22.453571 31.754145 153.75 0 0
1003_s_at 1 0.34723127 0.49105915 1 A 383.75 133.25 188.44395 383.75 0 0
1004_at 1 0.19441573 0.27494535 1 A 290.1 56.400005 79.76165 290.1 0 0
1005_at 1 0.31672025 0.44791007 1 A 124.4 39.4 55.720016 124.4 0 0
1006_at 1 0.45218188 0.6394817 1 A 31.4 14.198511 20.079727 31.4 0 0
1007_s_at 1 0.115599 0.16348167 1 P 5313.0996 614.189 868.5944 5313.0996 0 0
1008_f_at 1 0.11565181 0.16355635 1 P 3158.7 365.30933 516.62537 3158.7 0 0
1009_at 1 0.11557825 0.16345233 1 P 10017.45 1157.7993 1637.3755 10017.45 0 0
100_g_at 0.99999994 0.11583685 0.16381805 0.99999964 "P,M" 1747.25 202.39594 286.23108 1747.25 0 0
1010_at 1 0.490441 0.6935883 1 A 28.8 14.124701 19.975342 28.8 0 0
1011_s_at 1 0.11569888 0.16362293 1 P 2515.55 291.04633 411.60165 2515.55 0 0
1012_at 1 0.7375746 1.043088 1 A 25.15 18.550001 26.233664 25.15 0 0
1013_at 1 0.12547034 0.17744185 1 P 281 35.257168 49.861164 281 0 0
1014_at 1 0.116022974 0.16408126 1 "P,M" 1340.4 155.5172 219.93452 1340.4 0 0
1015_s_at 1 0.12713481 0.17979577 1 A 259.1 32.94063 46.585087 259.1 0 0
1016_s_at 1 0.6196069 0.87625647 1 A 22.55 13.972136 19.759583 22.55 0 0
1017_at 1 0.13011862 0.18401551 1 P 229.6 29.875237 42.249966 229.6 0 0
1018_at 1 0.12448671 0.17605078 1 A 296.7 36.935207 52.234272 296.7 0 0

Total number of rows: 12625

Table truncated, full table size 952 Kbytes.




Table 2: Cryptosporidium parvum-infected HCT-8 cells, 6 hours post-infection (average of 3 replicates) header descriptions
ID_REF
Ratio
StdErr Norm
StdDev Norm
t-test P-value
Call
VALUE
StdErr Raw
StdDev Raw
Control
StdErr Control
StdDev Control

Data table
ID_REF Ratio StdErr Norm StdDev Norm t-test P-value Call VALUE StdErr Raw StdDev Raw Control StdErr Control StdDev Control
1000_at 0.7424939 0.24907781 0.4314154 0.37724572 P 3017.8667 1012.3769 1753.4882 4064.5 0 0
1001_at 1.4793936 0.46788597 0.8104023 0.360888 A 115.46667 36.518505 63.251904 78.05 0 0
1002_f_at 0.50319785 0.26046336 0.45113575 0.15251903 A 77.36667 40.04624 69.36212 153.75 0 0
1003_s_at 0.31991312 0.25092635 0.43461716 0.07314563 A 122.76666 96.29299 166.78435 383.75 0 0
1004_at 1.052166 0.3611647 0.6255556 0.8910789 A 305.2333 104.773865 181.47365 290.1 0 0
1005_at 2.7990353 0.41553834 0.7197335 0.01026656 A 348.2 51.692974 89.53486 124.4 0 0
1006_at 0.93418264 0.44869065 0.777155 0.8926815 A 29.333334 14.088887 24.402668 31.4 0 0
1007_s_at 0.8989919 0.24907091 0.43140346 0.7122701 P 4776.433 1323.3385 2292.0896 5313.0996 0 0
1008_f_at 1.1775731 0.24908878 0.43143442 0.5273613 P 3719.5999 786.7967 1362.7719 3158.7 0 0
1009_at 1.3623095 0.24906388 0.4313913 0.24174719 P 13646.866 2494.9849 4321.4404 10017.45 0 0
100_g_at 0.85935044 0.24915142 0.4315429 0.6118581 P 1501.5 435.32983 754.01337 1747.25 0 0
1010_at 2.8877318 1.5535138 2.6907647 0.3294698 A 83.166664 44.74119 77.49402 28.8 0 0
1011_s_at 1.2897112 0.33835474 0.5860476 0.43149924 P 3244.3333 851.1483 1474.232 2515.55 0 0
1012_at 1.6580516 1.0008699 1.7335575 0.5516145 A 41.7 25.171877 43.59897 25.15 0 0
1013_at 0.7188612 0.2525287 0.43739253 0.34675512 P 202 70.96056 122.9073 281 0 0
1014_at 0.9372078 0.24921454 0.43165225 0.8173468 "P,A" 1256.2333 334.04718 578.5867 1340.4 0 0
1015_s_at 1.2636048 0.25313473 0.4384422 0.37425056 A 327.4 65.587204 113.600365 259.1 0 0
1016_s_at 3.53289 1.2113882 2.098186 0.123035334 A 79.666664 27.316803 47.31409 22.55 0 0
1017_at 1.5445703 0.26521668 0.45936877 0.11896251 P 354.63333 60.89374 105.471054 229.6 0 0
1018_at 0.6694753 0.25217363 0.43677753 0.28126737 A 198.63333 74.81992 129.5919 296.7 0 0

Total number of rows: 12625

Table truncated, full table size 1143 Kbytes.




Table 3: Cryptosporidium parvum-infected HCT-8 cells, 12 hours post-infection (average of 3 replicates) header descriptions
ID_REF
Ratio
StdErr Norm
StdDev Norm
t-test P-value
Call
VALUE
StdErr Raw
StdDev Raw
Control
StdErr Control
StdDev Control

Data table
ID_REF Ratio StdErr Norm StdDev Norm t-test P-value Call VALUE StdErr Raw StdDev Raw Control StdErr Control StdDev Control
1000_at 0.6494772 0.23628142 0.40925142 0.23458269 P 2639.8 960.3659 1663.4025 4064.5 0 0
1001_at 0.9404228 0.46434116 0.80426246 0.9040162 A 73.399994 36.24183 62.77269 78.05 0 0
1002_f_at 0.4327371 0.24830619 0.43007892 0.106490515 A 66.53333 38.177074 66.12463 153.75 0 0
1003_s_at 0.4373507 0.23823784 0.41264004 0.09924579 A 167.83334 91.42377 158.3506 383.75 0 0
1004_at 1.1791337 0.23970744 0.41518545 0.509123 A 342.06668 69.53914 120.44532 290.1 0 0
1005_at 7.0656486 0.91179264 1.5792712 0.01437779 "P,M" 878.9667 113.426994 196.46132 124.4 0 0
1006_at 0.37791932 0.4424119 0.7662799 0.25436932 A 11.866667 13.891733 24.061188 31.4 0 0
1007_s_at 0.8934458 0.2362741 0.40923873 0.68260866 P 4746.9663 1255.3479 2174.3264 5313.0996 0 0
1008_f_at 1.0848873 0.23629302 0.4092715 0.74320865 P 3426.8333 746.3787 1292.7659 3158.7 0 0
1009_at 1.1083261 0.23626666 0.40922585 0.67777467 P 11102.601 2366.7896 4099.4 10017.45 0 0
100_g_at 1.136815 0.23635936 0.4093864 0.60329294 P 1986.3 412.97888 715.3004 1747.25 0 0
1010_at 1.3472223 0.47130635 0.81632656 0.51467437 A 38.8 13.573623 23.510204 28.8 0 0
1011_s_at 1.708016 0.23630989 0.4093007 0.05784686 P 4296.6 594.4494 1029.6166 2515.55 0 0
1012_at 1.8515574 0.52335954 0.9064853 0.20219491 A 46.566666 13.162492 22.798103 25.15 0 0
1013_at 1.3264531 0.23993237 0.41557506 0.2668403 P 372.73334 67.421 116.77659 281 0 0
1014_at 1.1104646 0.23642617 0.40950215 0.67218053 P 1488.4667 316.90567 548.8967 1340.4 0 0
1015_s_at 1.2104721 0.24057294 0.41668457 0.44603676 A 313.63333 62.332455 107.962975 259.1 0 0
1016_s_at 2.7494457 0.61812836 1.0706297 0.048355255 A 62 13.938794 24.142698 22.55 0 0
1017_at 1.1001742 0.24173805 0.41870257 0.70643514 P 252.59999 55.50305 96.1341 229.6 0 0
1018_at 0.93315357 0.239557 0.4149249 0.7983433 A 276.86667 71.07656 123.108215 296.7 0 0

Total number of rows: 12625

Table truncated, full table size 1144 Kbytes.




Table 4: Cryptosporidium parvum-infected HCT-8 cells, 24 hours post-infection (average of 3 replicates) header descriptions
ID_REF
Ratio
StdErr Norm
StdDev Norm
t-test P-value
Call
VALUE
StdErr Raw
StdDev Raw
Control
StdErr Control
StdDev Control

Data table
ID_REF Ratio StdErr Norm StdDev Norm t-test P-value Call VALUE StdErr Raw StdDev Raw Control StdErr Control StdDev Control
1000_at 0.6147948 0.23852286 0.4131337 0.20473023 P 2498.8335 969.47614 1679.1819 4064.5 0 0
1001_at 1.0877641 0.32591203 0.56449616 0.80036086 A 84.9 25.43744 44.05894 78.05 0 0
1002_f_at 0.42319784 0.25377968 0.43955928 0.10764224 A 65.066666 39.018627 67.582245 153.75 0 0
1003_s_at 0.47157437 0.24101824 0.41745582 0.11598572 A 180.96667 92.49074 160.19865 383.75 0 0
1004_at 1.1841894 0.2428892 0.4206964 0.503389 A 343.53336 70.46216 122.04404 290.1 0 0
1005_at 3.0316186 0.29322863 0.5078869 0.001714396 A 377.13333 36.477634 63.181114 124.4 0 0
1006_at 0.3163482 0.48705372 0.84360176 0.25502843 A 9.933333 15.293487 26.489096 31.4 0 0
1007_s_at 0.8889538 0.23851348 0.41311747 0.6732352 P 4723.1 1267.246 2194.9343 5313.0996 0 0
1008_f_at 1.5486118 0.23853765 0.41315934 0.10499839 P 4891.5996 753.4689 1305.0464 3158.7 0 0
1009_at 1.1165749 0.238504 0.41310102 0.6585028 P 11185.233 2389.202 4138.219 10017.45 0 0
100_g_at 1.0448991 0.23862235 0.41330603 0.86275965 "P,A" 1825.7001 416.93292 722.149 1747.25 0 0
1010_at 1.6967593 0.5208189 0.90208477 0.27332932 A 48.86667 14.999583 25.98004 28.8 0 0
1011_s_at 1.3289075 0.23855917 0.4131966 0.26179788 P 3342.9333 600.10754 1039.4167 2515.55 0 0
1012_at 0.99138504 0.58136916 1.0069609 0.98910743 A 24.933332 14.621435 25.325068 25.15 0 0
1013_at 0.7534994 0.2431753 0.42119196 0.3853864 P 211.73332 68.33225 118.354935 281 0 0
1014_at 1.1907889 0.23870766 0.4134538 0.48256987 P 1596.1333 319.96375 554.1935 1340.4 0 0
1015_s_at 1.0272739 0.2439897 0.42260256 0.91805506 A 266.16666 63.217735 109.49633 259.1 0 0
1016_s_at 1.5816705 1.0091863 1.7479619 0.59331995 A 35.666668 22.757149 39.41654 22.55 0 0
1017_at 1.4335077 0.24546954 0.4251657 0.17556566 P 329.13333 56.35981 97.61805 229.6 0 0
1018_at 1.215706 0.24269778 0.42036486 0.43958986 A 360.69998 72.00843 124.72226 296.7 0 0

Total number of rows: 12625

Table truncated, full table size 1143 Kbytes.




Table 5: Cryptosporidium parvum-infected HCT-8 cells, 48 hours post-infection (average of 3 replicates) header descriptions
ID_REF
Ratio
StdErr Norm
StdDev Norm
t-test P-value
Call
VALUE
StdErr Raw
StdDev Raw
Control
StdErr Control
StdDev Control

Data table
ID_REF Ratio StdErr Norm StdDev Norm t-test P-value Call VALUE StdErr Raw StdDev Raw Control StdErr Control StdDev Control
1000_at 0.7317751 0.24743968 0.4285781 0.35770527 P 2974.3 1005.7185 1741.9556 4064.5 0 0
1001_at 1.7838993 1.007355 1.74479 0.5036032 A 139.23334 78.624054 136.18085 78.05 0 0
1002_f_at 0.28422764 0.27155042 0.47033912 0.07793128 A 43.7 41.75088 72.314644 153.75 0 0
1003_s_at 0.35127035 0.25143588 0.4354997 0.08177273 A 134.8 96.48851 167.123 383.75 0 0
1004_at 1.5322303 0.25441763 0.44066426 0.12753898 A 444.50003 73.80656 127.8367 290.1 0 0
1005_at 4.9032693 0.55305237 0.95791477 0.003859473 A 609.9667 68.79972 119.16461 124.4 0 0
1006_at 0.5021232 0.601375 1.041612 0.46843955 A 15.766667 18.883175 32.70662 31.4 0 0
1007_s_at 0.81706226 0.24742466 0.4285521 0.51328206 P 4341.1333 1314.5918 2276.9397 5313.0996 0 0
1008_f_at 1.5596291 0.50646365 0.87722075 0.3444274 P 4926.4 1599.7667 2770.8772 3158.7 0 0
1009_at 1.0445873 0.2474094 0.42852566 0.8684688 P 10464.101 2478.4114 4292.7344 10017.45 0 0
100_g_at 0.85610723 0.24759948 0.42885488 0.601921 A 1495.8334 432.61816 749.31665 1747.25 0 0
1010_at 2.4409723 1.2049878 2.0871 0.30532688 A 70.3 34.703648 60.10848 28.8 0 0
1011_s_at 0.94679093 0.24749804 0.42867917 0.84356236 P 2381.7 622.5937 1078.3639 2515.55 0 0
1012_at 1.9854207 0.7276895 1.2603952 0.26865363 A 49.933334 18.301392 31.69894 25.15 0 0
1013_at 1.1083037 0.25487253 0.44145218 0.6995218 P 311.43335 71.61918 124.04806 281 0 0
1014_at 1.0695564 0.24773642 0.42909208 0.79713845 P 1433.6333 332.06592 575.155 1340.4 0 0
1015_s_at 0.5367297 0.25616595 0.44369245 0.16824657 A 139.06667 66.3726 114.96071 259.1 0 0
1016_s_at 1.6215818 0.80234206 1.3896972 0.49496788 A 36.566666 18.092812 31.337667 22.55 0 0
1017_at 1.6559234 0.25851056 0.44775343 0.08487848 P 380.2 59.354023 102.804184 229.6 0 0
1018_at 0.82979435 0.2541131 0.44013682 0.55092764 A 246.2 75.39536 130.5886 296.7 0 0

Total number of rows: 12625

Table truncated, full table size 1142 Kbytes.




Table 6: Cryptosporidium parvum-infected HCT-8 cells, 72 hours post-infection (average of 3 replicates) header descriptions
ID_REF
Ratio
StdErr Norm
StdDev Norm
t-test P-value
Call
VALUE
StdErr Raw
StdDev Raw
Control
StdErr Control
StdDev Control

Data table
ID_REF Ratio StdErr Norm StdDev Norm t-test P-value Call VALUE StdErr Raw StdDev Raw Control StdErr Control StdDev Control
1000_at 0.79978675 0.21222512 0.3675847 0.4150743 P 3250.7332 862.589 1494.048 4064.5 0 0
1001_at 1.0574418 0.3773944 0.65366626 0.8851101 A 82.53333 29.455639 51.01866 78.05 0 0
1002_f_at 0.44531164 0.24124493 0.41784847 0.10505821 A 68.46667 37.09141 64.24421 153.75 0 0
1003_s_at 0.47826278 0.21710843 0.3760428 0.0956061 A 183.53334 83.31536 144.30644 383.75 0 0
1004_at 1.3162129 0.22073135 0.3823179 0.24740808 A 381.83334 64.03417 110.91044 290.1 0 0
1005_at 3.6623793 0.46650764 0.8080149 0.006215178 A 455.6 58.033554 100.51706 124.4 0 0
1006_at 0.5785563 0.60081905 1.040649 0.5335406 A 18.166666 18.865719 32.676384 31.4 0 0
1007_s_at 0.8123632 0.21220668 0.36755273 0.441698 P 4316.1665 1127.4752 1952.8444 5313.0996 0 0
1008_f_at 1.6041832 0.32183674 0.5574376 0.12586096 P 5067.1333 1016.58563 1760.778 3158.7 0 0
1009_at 1.1570476 0.212188 0.3675204 0.51287884 P 11590.666 2125.5828 3681.6172 10017.45 0 0
100_g_at 0.96780646 0.212421 0.36792395 0.8891556 A 1690.9999 371.1526 642.8551 1747.25 0 0
1010_at 1.420139 0.64854264 1.1233088 0.56326044 A 40.9 18.678028 32.35129 28.8 0 0
1011_s_at 1.5567569 0.38665533 0.66970664 0.2298899 P 3916.0999 972.6509 1684.6807 2515.55 0 0
1012_at 1.2882704 0.7331682 1.2698845 0.7204585 A 32.4 18.439178 31.937593 25.15 0 0
1013_at 0.9093713 0.22128256 0.38327262 0.709616 P 255.53334 62.180393 107.6996 281 0 0
1014_at 1.147717 0.21258889 0.36821476 0.53713113 P 1538.3999 284.95413 493.55502 1340.4 0 0
1015_s_at 1.1125691 0.23604393 0.40884006 0.6602636 A 288.26666 61.15898 105.93046 259.1 0 0
1016_s_at 3.0273468 0.8109602 1.4046242 0.08771176 A 68.26666 18.28715 31.67427 22.55 0 0
1017_at 1.4242162 0.46152276 0.79938084 0.42118314 P 327 105.96562 183.53784 229.6 0 0
1018_at 0.61139196 0.22036214 0.38167843 0.17601466 A 181.4 65.381454 113.243996 296.7 0 0

Total number of rows: 12625

Table truncated, full table size 1142 Kbytes.




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