Affymetrix GeneChip Mouse Expression Array MOE430A [MOE430A]

Impaired maturation but normal proliferative activity of myeloid progenitor cells in mice lacking the novel Polycomb group protein MBT-1

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Gene expression in the fetal liver cells obtained from MBT-1+/+ or MBT-1-/- embryos at E14.5 were analyzed using Affymetrix Genechip to assess the effect of the gene knock-out on hematopoietic cells.

Genotype

Transcription profiling of mouse fetal liver cells obtained from MBT-1+/+ or MBT-1-/- embryos at E14.5

Gene expression in the fetal liver cells obtained from MBT-1+/+ or MBT-1-/- embryos at E14.5 were analyzed using Affymetrix Genechip to assess the effect of the gene knock-out on hematopoietic cells.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4[]

Title: Affymetrix EukGE-WS2v4 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-6352

MBT-1+/+ or MBT-1-/- fetal liver cells were prepared from each genotype of embryo obtained by intercross breeding of MBT-1+/- mice.   Mice were maintained in SPF.

P-MEXP-6353

The fetal liver was excised from the embryo at E14.5 and single cell suspension was prepared.

P-MEXP-6355

1. Collect cells by centrifuge at 5000rpm for 5 min, carefully remove all supernatant. Loosen the cell pellet thoroughly by flicking the tube. 2. Disrupt cells by addition of Buffer RLT a. Aliquot certain amount of buffer RLT, add B-ME (add 10ul B-ME to 1ml buffer RLT) and mix well. b. Add appropriate volume of buffer RLT to loosened cells: For <5x106 cells, add 350ul buffer RLT For 5x106-1x107 cells, add 600ul buffer RLT Do not use more than 1x107 c. Vortex thoroughly for 30sec, pipet up and down 10 times to disrupt the cells. 3. Add 1 volume (300ul or 600ul) of 70% ethanol to the homogenized lysate and mix well by pipetting. Do not centrifuge. 4. Apply up to 700ul of the sample, including any precipitate that may have formed to an Rneasy mini column placed in a 2ml collection tube and centrifuge for 1min at 10,000rpm, reload the flow-through to the same column and centrifuge up to three times. Discard the flow-through. If the volume exceeds 700ul, load aliquots successively onto the Rneasy column and centrifuge as above. 5. Add 700ul buffer RW1 to the Rneasy column and centrifuge for 15s at 10,000rpm to wash the column, discard the flow-through. 6. Pipet 500ul buffer RPE (ensure Ethanol is added to buffer RPE before use) onto the Rneasy column and centrifuge for 1min at 10,000rpm to wash the column. Discard the flow-through. 7. Repeat step 6 to wash one more time. 8. Leave the column cap open and centrifuge at full speed for 2 min to eliminate any buffer RPE completely. 9. To elute, pre-warm the Rnase-free water to 65C, tranfer the Rneasy column to a new 1.5ml collection tube, pipet 25ul pre-warmed water directly onto the Rneasy silica-gel membrane. Close the tube and stand at room temperature for 2min. Centrifuge for 1min at 12,000rpm to elute. 10. Apply another 25ul of pre-warmed water to the column, stand 2min and centrifuge at 12,000rpm for 1min. Now you have about 50ul of elution buffer in the collection tube. 11. Pipet the 50ul of eluted buffer to the same column, stand at room temperature for 2min and centrifuge at 12,000rpm for 2min 12. Optional: you can vacuum dry to bring the volume to 20ul if the RNA concentration is too low. 13. Take 1ul of RNA, dilute 60 times and measure the concentration and ratio of OD260/280. 

P-MEXP-6356

TRANPRTCL6360

MAS 5.0 software (Affymetrix, Inc) was used to normalize the data. The control sample was used as the baseline. The resulting fold change (shown as log base 2) shows the expression level change for each gene (Please see the KO vs WT.CHP file)

Transcription profiling of mouse fetal liver cells obtained from MBT-1+/+ or MBT-1-/- embryos at E14.5

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