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Sample GSM575137 Query DataSets for GSM575137
Status Public on Aug 05, 2010
Title PP/5F2
Sample type RNA
 
Channel 1
Source name Bombyx 5th feeding stage fat body
Organism Bombyx mori
Characteristics tissue: fat body
Treatment protocol Fat body was vivisected in PBS and fat body quickly frozen in liquid nitrogen.
Growth protocol Bombyx larvae (Nistari) were provided by The Sericultural Research Institute, Chinese Academy of Agricultural Sciences. They were reared with fresh mulberry leaves in the laboratory at 25oC under 14 hour light/10 hour dark cycles.
Extracted molecule total RNA
Extraction protocol the different stages Bombyx fat bodys were dissected and quickly frozen in liquid nitrogen, total RNA was extracted with Trizol Reagent (BD Biosciences Clontech, Palo Alto, CA) and ethanol precipitation
Label Cy3
Label protocol 2 μg amplified RNA was mixed with 4 μg random nanomer, denatured at 65℃ for 5min, and cooled on ice. Then, 5 μL of 4×first-strand buffer, 2 μL of 0.1M DTT, and 1.5 μL CbcScript Ⅱ reverse transcriptase were added. The mixtures were incubated at 25℃ for 10 min, then at 37℃ for 90 min. The cDNA products were purified using a PCR NucleoSpin Extract II Kit (MN) and vacuum evaporated to 14 μL. The cDNA was mixed with 4 μg random nanomer, heated to 95℃ for 3 min, and snap cooled on ice for 5 min. Then, 5 μL Klenow buffer, dNTP, and Cy5-dCTP or Cy3-dCTP (GE Healthcare) were added to final concentrations of 240 μM dATP, 240 μM dGTP, 240 μM dTTP, 120 μM dCTP, and 40 μM Cy-dCTP. 1.2 μL Klenow enzyme was then added, and the reaction was performed at 37℃ for 90 min. Labeled cDNA was purified with a PCR NucleoSpin Extract II Kit (MN) and resuspended in elution buffer. Labeled controls and test samples were quantitatively adjusted based on the efficiency of Cy5-dCTP or Cy3-dCTP incorporation and dissolved in 80 μl hybridization solution containing 3×SSC, 0.2% SDS, 5×Denhardt’s solution and 25% formamide. DNA in hybridization solution was denatured at 95℃ for 3 min prior to loading onto a microarray
 
Channel 2
Source name Bombyx prepupa fat body
Organism Bombyx mori
Characteristics tissue: fat body
Treatment protocol Fat body was vivisected in PBS and fat body quickly frozen in liquid nitrogen.
Growth protocol Bombyx larvae (Nistari) were provided by The Sericultural Research Institute, Chinese Academy of Agricultural Sciences. They were reared with fresh mulberry leaves in the laboratory at 25oC under 14 hour light/10 hour dark cycles.
Extracted molecule total RNA
Extraction protocol the different stages Bombyx fat bodys were dissected and quickly frozen in liquid nitrogen, total RNA was extracted with Trizol Reagent (BD Biosciences Clontech, Palo Alto, CA) and ethanol precipitation
Label Cy5
Label protocol 2 μg amplified RNA was mixed with 4 μg random nanomer, denatured at 65℃ for 5min, and cooled on ice. Then, 5 μL of 4×first-strand buffer, 2 μL of 0.1M DTT, and 1.5 μL CbcScript Ⅱ reverse transcriptase were added. The mixtures were incubated at 25℃ for 10 min, then at 37℃ for 90 min. The cDNA products were purified using a PCR NucleoSpin Extract II Kit (MN) and vacuum evaporated to 14 μL. The cDNA was mixed with 4 μg random nanomer, heated to 95℃ for 3 min, and snap cooled on ice for 5 min. Then, 5 μL Klenow buffer, dNTP, and Cy5-dCTP or Cy3-dCTP (GE Healthcare) were added to final concentrations of 240 μM dATP, 240 μM dGTP, 240 μM dTTP, 120 μM dCTP, and 40 μM Cy-dCTP. 1.2 μL Klenow enzyme was then added, and the reaction was performed at 37℃ for 90 min. Labeled cDNA was purified with a PCR NucleoSpin Extract II Kit (MN) and resuspended in elution buffer. Labeled controls and test samples were quantitatively adjusted based on the efficiency of Cy5-dCTP or Cy3-dCTP incorporation and dissolved in 80 μl hybridization solution containing 3×SSC, 0.2% SDS, 5×Denhardt’s solution and 25% formamide. DNA in hybridization solution was denatured at 95℃ for 3 min prior to loading onto a microarray
 
 
Hybridization protocol Arrays were hybridized was preformed in a CapitalBio BioMixerTM II Hybridization Station overnight at a rotation speed of 8 rpm and a temperature of 42℃ and washed with two consecutive solutions (0.2% SDS, 2× SSC at 42℃ for 5 min, and 0.2× SSC for 5 min at room temperature).
Scan protocol confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software (Both from CapitalBio)
Description prepupal compared to 5th instar feeding 2, Biological replicate 2 of 3 PP compared to 5F
Data processing LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used.
 
Submission date Aug 04, 2010
Last update date Aug 04, 2010
Contact name Ling Tian
E-mail(s) tianling80@yahoo.com.cn, shengli@sippe.ac.cn
Phone (86)021-54924163
Fax (86)021-54924163
Organization name Chinese Academy of Sciences
Department Institute of Plant Physiology and Ecology
Lab Key Laboratory of Developmental and Evolutionary Biology
Street address Fenglin Road 300
City Shanghai
ZIP/Postal code 200032
Country China
 
Platform ID GPL10763
Series (1)
GSE23424 Differentially expressed genes at molting and metamorphosis stages in Bombyx fat body

Data table header descriptions
ID_REF
VALUE normalized log2 ratio (Cy5/Cy3) representing test/reference

Data table
ID_REF VALUE
23857 -1.608672193
23761 1.026162654
23809 0.610038557
23665 -0.690358127
23713 -0.25239777
23617 -11.70274988
23041 0.05338942
23905 2.622719552
23953 2.402503901
24001 2.036573665
24049 1.867461614
24097 -1.196607621
24145 4.473241415
24193 0.90180335
24241 3.235711745
12 -0.119510373
24 -0.26760054
108 0.459011864
120 -0.258080052
204 -0.20572971

Total number of rows: 24288

Table truncated, full table size 425 Kbytes.




Supplementary file Size Download File type/resource
GSM575137.lsr.gz 1.4 Mb (ftp)(http) LSR
Processed data included within Sample table

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