fetal liver cells were dissected in the liver perfusion medium (Invitrogen), and a single cell suspension was obtained by collagenase digestion. Using anti-Dlk antibody to isolate Dlk postive cells (hepatoblast) and collect the cells by MACS Separator.
Growth protocol
mice growth in 25°C
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Trizol following manufacturer's instructions
Label
cy3
Label protocol
Isolated mRNA is reverse transcribed with 5’ Cy3 or Cy5 labeled random 9-mers (Operon Technologies, Inc., Alameda, CA). Reactions are incubated for 2 hrs. at 37°C with 200 ng polyA RNA, 200 Units M-MLV reverse transcriptase (Life Technologies, Gaithersburg, MD), 4 mM DTT, 1 unit RNase Inhibitor (Ambion, Austin, TX), 0.5 mM dNTPs, and 2 mg labeled 9-mers in 25 mL volume with enzyme buffer supplied by the manufacturer. The reactions is terminated by incubation at 85°C for 5 min. The paired reactions are combined and purified with a TE-30 column (Clontech, Palo Alto, CA), brought to 90 mL with dH2O, and precipitated with 2 mL 1mg/mL glycogen, 60 mL 5M NH4OAc, and 300 mL EtOH. After centrifugation the supernatant is decanted and the pellet is resuspended in 24 mL of hybridization buffer: 5XSSC, 0.2% SDS, 1 mM DTT.
fetal liver cells were dissected in the liver perfusion medium (Invitrogen), and a single cell suspension was obtained by collagenase digestion. Using anti-Dlk antibody to isolate Dlk postive cells (hepatoblast) and collect the cells by MACS Separator.
Growth protocol
mice growth in 25°C
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Trizol following manufacturer's instructions
Label
cy5
Label protocol
Isolated mRNA is reverse transcribed with 5’ Cy3 or Cy5 labeled random 9-mers (Operon Technologies, Inc., Alameda, CA). Reactions are incubated for 2 hrs. at 37°C with 200 ng polyA RNA, 200 Units M-MLV reverse transcriptase (Life Technologies, Gaithersburg, MD), 4 mM DTT, 1 unit RNase Inhibitor (Ambion, Austin, TX), 0.5 mM dNTPs, and 2 mg labeled 9-mers in 25 mL volume with enzyme buffer supplied by the manufacturer. The reactions is terminated by incubation at 85°C for 5 min. The paired reactions are combined and purified with a TE-30 column (Clontech, Palo Alto, CA), brought to 90 mL with dH2O, and precipitated with 2 mL 1mg/mL glycogen, 60 mL 5M NH4OAc, and 300 mL EtOH. After centrifugation the supernatant is decanted and the pellet is resuspended in 24 mL of hybridization buffer: 5XSSC, 0.2% SDS, 1 mM DTT.
Hybridization protocol
Probe solutions are thoroughly resuspended by incubating at 65°C for 5 min. with mixing. The probe is applied to the array and covered with a 22 mm2 glass cover-slip, and placed in a sealed chamber to prevent evaporation. After hybridization at 60°C for 6.5 hours, the slide is washed in three consecutive washes of decreasing ionic strength.
Scan protocol
The microarray is scanned in both Cy3 and Cy5 channels with Axon GenePix scanners (Foster City, CA) with a 10 mm resolution. The signal is converted into 16-bits per-pixel resolution, yielding a 65,536 count dynamic range.
Description
Isolate hepatoblast by ant-Dlk antibody though MACS
Data processing
Incyte GEMtool software (Incyte Pharmaceuticals, Inc., Palo Alto, CA) was used for image analysis. The element are determined by a gridding and region detection algorithm. The area surrounding each element image is used to calculate a local background and is subtracted from the total element signal. Background subtracted element signals are used to calculate Cy3:Cy5 ratios. The average of the resulting total Cy3 and Cy5 signal gives a ratio that is used to balance or normalize the signals.
log2 ratio of PRE_VALUE: Cy3/Cy5 ratio (Unbalanced differential expression for the element. If P1Signal > P2Signal then +(P1Signal / P2Signal). IF P1Signal < P2Signal then -(P2Signal/ P1Signal).)
PRE_VALUE
Cy3/Cy5 ratio (Unbalanced differential expression for the element. If P1Signal > P2Signal then +(P1Signal / P2Signal). IF P1Signal < P2Signal then -(P2Signal/ P1Signal).)
