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BioAssay: AID 1904

siRNA Circadian Assay

To identify additional clock genes and modifiers, we conducted a genome-wide screen of ~90,000 siRNAs (QIAGEN Inc) in a human cellular model of clock function. Knockdown of nearly a thousand genes reduced ..more
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 Tested Substances
 Tested Substances
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Active(527)
 
 
Inconclusive(97)
 
 
Unspecified(32740)
 
 
 Related BioAssays
 Related BioAssays
AID: 1904
Data Source: The Genomics Institute of the Novartis Research Foundation (GNF) (siRNA_circadian)
Depositor Category: Literature, Author
BioAssay Version:
Deposit Date: 2009-08-19
Hold-until Date: 2009-09-24
Modify Date: 2010-07-01

Data Table ( Complete ):           Active    All
Depositor Specified Assays
AIDNameTypeComment
2774LOPAC Circadian Assayscreening
493019Longdaysin Circadian Assayconfirmatory
Description:
Description:

To identify additional clock genes and modifiers, we conducted a genome-wide screen of ~90,000 siRNAs (QIAGEN Inc) in a human cellular model of clock function. Knockdown of nearly a thousand genes reduced
rhythm amplitude; these are presumably due to toxic effects on cells. Hundreds of genes potently modified period length or increased amplitude.
Protocol
Cell Culture Growth Conditions: U2OS cells were obtained from the American Type Culture Collection (ATCC), and were grown in regular DMEM supplemented with 10% FBS and antibiotics. Lentiviral
Bmal1-dLuc reporter was introduced into U2OS cells via lentivirus-mediated infection as described (Liu et al., PLoS Genetics, 2008). Stable cell lines were selected with blasticidin and clonal lines were sorted by FACS-based single cell sorting in 96-well plates. The clonal lines are genetically and morphologically indistinguishable from parental cells, and represent the average period length of the infected cell population.

Transfection: Human genome-wide siRNA set against 22,468 known or predicted genes was purchased from Qiagen Inc, and pre-spotted into 384-well plates with 2 siRNA/well, 2 well/gene in duplicates, and 294 plates in total. Subsequently, U2OS circadian reporter cells were reverse transfected with siRNAs in 384-well plates. Briefly, we trypsinized rapidly growing cells and re-suspended them in DMEM containing 20% FBS without antibiotics at 0.1X106 cells/ml. We next added 20 ul of transfection reagent mixture (3.3 ul/ml or 0.066 ul/well Lipofectamine 2000 in Opti-MEM) to each well containing pre-spotted siRNA constructs (1 pmol; 0.5 pmol for each siRNA; final concentration of 12.5 nM), incubated at room temperature for 20 min, and added 20 ul of cells (2000 cells/well) with our robotic system. Approximately 18 hr after transfection, we replaced this media with 60 ul pre-warmed fresh DMEM containing 10% FBS and antibiotics and allow the cells to grow for an additional two days.

Luminescence recording: Three days post-transfection, we replaced this media with 60 ul HEPES-buffered explant medium supplemented with luciferin (1 uM) (Promega) and B-27 supplements (Invitrogen), and the plates were sealed with an optically clear film (USA Scientific). We next loaded these plates in a 36oC incubator and recorded bioluminescence expression with a ViewLux (Perkin Elmer). We measured bioluminescence for 30 seconds every two hours for four days.
Comment
The data contain plate and well position and bioluminescence value from the primary screen. X-axis and Y-axis indicate time-course (from 0 to 78h) and plate/well position, respectively. Time 0 is set up as 8h after the change of luciferin-containing-medium /recording starts. Bioluminescence data was detrended by subtracting a best fit line (first order polynomial).
Result Definitions
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TIDNameDescriptionAnnotationHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Hit ConfirmationConfirmed by individual siRNA in Bmal1_dLucString
2PhenotypeBiological effect type.String
3Gene Target IDGene ID in NCBI Gene DatabaseString
4Gene SymbolGene Symbol in NCBI Gene DatabaseTarget nameString
5Nucleotide AccessionAccession number of NCBI Nucleotide DatabaseString
6RNAi Probe IDRNAi Probe ID in NCBI Probe DatabaseString
7GNFid of siRNA 1String
8GNFid of siRNA 2String
9Plate of Replicate 1String
10Plate of Replicate 2String
11Virtual Plate numberString
12Well numberString
13replicate1_readout_0_hrbioluminescence measurementFloat
14replicate1_readout_2_hrbioluminescence measurementFloat
15replicate1_readout_4_hrbioluminescence measurementFloat
16replicate1_readout_6_hrbioluminescence measurementFloat
17replicate1_readout_8_hrbioluminescence measurementFloat
18replicate1_readout_10_hrbioluminescence measurementFloat
19replicate1_readout_12_hrbioluminescence measurementFloat
20replicate1_readout_14_hrbioluminescence measurementFloat
21replicate1_readout_16_hrbioluminescence measurementFloat
22replicate1_readout_18_hrbioluminescence measurementFloat
23replicate1_readout_20_hrbioluminescence measurementFloat
24replicate1_readout_22_hrbioluminescence measurementFloat
25replicate1_readout_24_hrbioluminescence measurementFloat
26replicate1_readout_26_hrbioluminescence measurementFloat
27replicate1_readout_28_hrbioluminescence measurementFloat
28replicate1_readout_30_hrbioluminescence measurementFloat
29replicate1_readout_32_hrbioluminescence measurementFloat
30replicate1_readout_34_hrbioluminescence measurementFloat
31replicate1_readout_36_hrbioluminescence measurementFloat
32replicate1_readout_38_hrbioluminescence measurementFloat
33replicate1_readout_40_hrbioluminescence measurementFloat
34replicate1_readout_42_hrbioluminescence measurementFloat
35replicate1_readout_44_hrbioluminescence measurementFloat
36replicate1_readout_46_hrbioluminescence measurementFloat
37replicate1_readout_48_hrbioluminescence measurementFloat
38replicate1_readout_50_hrbioluminescence measurementFloat
39replicate1_readout_52_hrbioluminescence measurementFloat
40replicate1_readout_54_hrbioluminescence measurementFloat
41replicate1_readout_56_hrbioluminescence measurementFloat
42replicate1_readout_58_hrbioluminescence measurementFloat
43replicate1_readout_60_hrbioluminescence measurementFloat
44replicate1_readout_62_hrbioluminescence measurementFloat
45replicate1_readout_64_hrbioluminescence measurementFloat
46replicate1_readout_66_hrbioluminescence measurementFloat
47replicate1_readout_68_hrbioluminescence measurementFloat
48replicate1_readout_70_hrbioluminescence measurementFloat
49replicate1_readout_72_hrbioluminescence measurementFloat
50replicate1_readout_74_hrbioluminescence measurementFloat
51replicate1_readout_76_hrbioluminescence measurementFloat
52replicate1_readout_78_hrbioluminescence measurementFloat
53replicate2_readout_0_hrbioluminescence measurementFloat
54replicate2_readout_2_hrbioluminescence measurementFloat
55replicate2_readout_4_hrbioluminescence measurementFloat
56replicate2_readout_6_hrbioluminescence measurementFloat
57replicate2_readout_8_hrbioluminescence measurementFloat
58replicate2_readout_10_hrbioluminescence measurementFloat
59replicate2_readout_12_hrbioluminescence measurementFloat
60replicate2_readout_14_hrbioluminescence measurementFloat
61replicate2_readout_16_hrbioluminescence measurementFloat
62replicate2_readout_18_hrbioluminescence measurementFloat
63replicate2_readout_20_hrbioluminescence measurementFloat
64replicate2_readout_22_hrbioluminescence measurementFloat
65replicate2_readout_24_hrbioluminescence measurementFloat
66replicate2_readout_26_hrbioluminescence measurementFloat
67replicate2_readout_28_hrbioluminescence measurementFloat
68replicate2_readout_30_hrbioluminescence measurementFloat
69replicate2_readout_32_hrbioluminescence measurementFloat
70replicate2_readout_34_hrbioluminescence measurementFloat
71replicate2_readout_36_hrbioluminescence measurementFloat
72replicate2_readout_38_hrbioluminescence measurementFloat
73replicate2_readout_40_hrbioluminescence measurementFloat
74replicate2_readout_42_hrbioluminescence measurementFloat
75replicate2_readout_44_hrbioluminescence measurementFloat
76replicate2_readout_46_hrbioluminescence measurementFloat
77replicate2_readout_48_hrbioluminescence measurementFloat
78replicate2_readout_50_hrbioluminescence measurementFloat
79replicate2_readout_52_hrbioluminescence measurementFloat
80replicate2_readout_54_hrbioluminescence measurementFloat
81replicate2_readout_56_hrbioluminescence measurementFloat
82replicate2_readout_58_hrbioluminescence measurementFloat
83replicate2_readout_60_hrbioluminescence measurementFloat
84replicate2_readout_62_hrbioluminescence measurementFloat
85replicate2_readout_64_hrbioluminescence measurementFloat
86replicate2_readout_66_hrbioluminescence measurementFloat
87replicate2_readout_68_hrbioluminescence measurementFloat
88replicate2_readout_70_hrbioluminescence measurementFloat
89replicate2_readout_72_hrbioluminescence measurementFloat
90replicate2_readout_74_hrbioluminescence measurementFloat
91replicate2_readout_76_hrbioluminescence measurementFloat
92replicate2_readout_78_hrbioluminescence measurementFloat

RNAi Target.
Additional Information
Substance Type: Nucleotide

Data Table (Concise)
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