THO2 and HPR1 proteins were co-depleted from Drosophila S2 cells and their role in mRNA export analysed by comparing total RNA and cytoplasmic RNA

RNAi knock down in Drosophila of THO2 and HPR1 proteins from S2 cells

Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster

Affymetrix GeneChip Drosophila Genome [DrosGenome1]

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siRNA treatment

THO2 and HPR1 proteins were co-depleted from Drosophila S2 cells and their role in mRNA export analysed by comparing total RNA and cytoplasmic RNA


Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-2869

Two different endogenous Drosophila proteins (potential export factors) have been inactivated using RNAi treatment. Cells were transfected with 20 microgramm dsRNA per 10E6 cells (day 0) and re-transfected on day 4. The effect on mRNA export is analyzed on day 8 by monitoring the levels of mRNAs in cytoplasmic and total samples. Samples from knock-downs are compared to samples obtained from (GFP transfected) control cells processed in parallel. The potential export factors targeted by RNAi are: HPR1 and THO2.

P-MEXP-2875

P-MEXP-2882

Total RNA was extracted with TRIzol (GIBCO/Invitrogen) accroding to the manufacturer's instructions and resuspended in ddH2O. The sample was brought to 40 mM Tris pH 7.9, 6 mM MgCl2, 10 mM NaCl, 2 mM spermidine, 10 mM DTT and treated with 2 units of RNase-free DNase I (Promega) for 30 min at 37 °C. Following phenol:chloroform:isoamylalcohol and choroform extractions, the RNA was ethanol-precipitated and resuspended in ddH2O. Two total RNA preparations were isolated from a single knockdown experiment. Equal quantities of these preparations were pooled with the equal quantities of two preparations isolated from an independent knock down. These pools of 4 total preparations from 2 independent knockdowns represent one total sample. 

P-MEXP-2883

P-MEXP-2887

Data analysis was done using the GeneSpring(TM) 4.2 software (Silicon Genetics). In short, unscaled data were exported from Microarray suite *.chp-files into Excel spreadsheets. In order not to exclude genes that are e.g. strongly down regulated in a knockdown experiment, expression data of genes detected as present in the corresponding EGFP knockdown and/or the knockdown experiment were then imported into GeneSpring (EGFP control knockdown = control channel, knockdown experiment = signal channel), intensity-dependent normalized and visualized with scatter-plots.

P-MEXP-2892

To reduce potentially insignificant variations in the preparation of the RNA, two total RNA preparations were isolated from a single knockdown experiment. These preparations were pooled with the equivalent preparations isolated from an independent knockdown, to minimize differences in knockdown efficiencies. This pool of 4  total RNA preparations from 2 independent knockdowns is referred to total sample.

P-MEXP-2893

To reduce potentially insignificant variations in the preparation of the RNA, three cytoplasmic RNA preparations were isolated from a single knockdown experiment. These preparations were pooled with the equivalent preparations isolated from an independent knockdown, to minimize differences in knockdown efficiencies. This pool of 6 cytoplasmic RNA preparations from 2 independent knockdowns is referred to as 'cytoplasmic samples'.

P-MEXP-3033

Standard Affymetrix protocols were used for labeling, as described in the ""GeneChip Expression Analysis Technical Manual"".

RNAi knock down in Drosophila of THO2 and HPR1 proteins from S2 cells

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