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
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
4th molting compared to 5th instar feeding 1, Biological replicate 1 of 3 4M compared to 5F
Data processing
LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used.
