strain/background: C57B6/J black × 129Sv cell type: mouse embryonic fibroblasts genotype: PSEN1&2-/- genetic modification: retroviral infection with human ARF6 (puromycin resistant)
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from 80% confluent monolayer of cells using TriPure reagent and chloroform extraction. (1) Homogenization: Culture cells in 10cm² dishes, Use 1ml TriPure reagent/10cm² dish (when appending too little TriPure reagent, the chances are higher to contaminate the RNA with genomic DNA), Pass cell lysates several times through a pipet, To reduce viscosity, shear the genomic DNA with 2 passes through a 26 gauge needle prior to the addition of chloroform, Let stand at RT until completely resolved ~5min, Vortex samples. (2) Phase separation: Add 0.2ml chloroform per 1ml TriPure reagent, Shake vigorously for 15sec by hand, Incubate 2-3min at RT, Spin 15min at 12,000rpm at 4°C, Three layers are formed (top layer = aqueous phase containing RNA; white interphase layer = containing DNA and protein; bottom layer = red organic phase containing DNA and protein). (3) RNA precipitation: Mix aqueous phase (top layer) with 0.5ml isopropanol per 0.7-0.75ml TriPure reagent (new eppe), Incubate 10min at RT, Spin 10min at 12,000rpm at 4°C, The RNA precipitate forms a gel-like pellet (often invisible before centrifugation) on the side and bottom of the tube. (4) RNA wash: Remove supernatant, Wash RNA pellet with at least 1ml 75% EtOH per 1ml TriPure reagent used for the initial RNA isolation step, Vortex, Spin 5min at 7,500xg at 4°C. (5) Redissolve the RNA: Air-dry RNA pellet (5-10min) (Do not let RNA pellet dry out completely as this will greatly decrease its solubility), Redisolve in RNAse free water, Pass solution a few times through a pipet tip and incubate 10min at 55°C-60°C to avoid problems with partially dissolved RNA, Expected yield for fibroblasts: 5-7µg from 1*106 cells. (6) Store RNA samples at -80°C (1µg needed for complete exp)
Label
Cy5
Label protocol
RNA concentration and purity were determined spectrophotometrically using the NanoDrop ND-1000 (NanoDrop Technologies), and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 100ng of total RNA spiked with 10 viral polyA transcript controls (Agilent) was converted to double-stranded cDNA in a reverse transcription reaction. Subsequently, the sample was converted to antisense cRNA, amplified and labeled with Cyanine 5-CTP (Cy5) in an in vitro transcription reaction according to the manufacturer's protocol (Agilent).
strain/background: C57B6/J black × 129Sv cell type: mouse embryonic fibroblasts genotype: PSEN1&2-/- genetic modification: retroviral infection with human PSEN1 D257A/D385A mutant (puromycin resistant)
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from 80% confluent monolayer of cells using TriPure reagent and chloroform extraction. (1) Homogenization: Culture cells in 10cm² dishes, Use 1ml TriPure reagent/10cm² dish (when appending too little TriPure reagent, the chances are higher to contaminate the RNA with genomic DNA), Pass cell lysates several times through a pipet, To reduce viscosity, shear the genomic DNA with 2 passes through a 26 gauge needle prior to the addition of chloroform, Let stand at RT until completely resolved ~5min, Vortex samples. (2) Phase separation: Add 0.2ml chloroform per 1ml TriPure reagent, Shake vigorously for 15sec by hand, Incubate 2-3min at RT, Spin 15min at 12,000rpm at 4°C, Three layers are formed (top layer = aqueous phase containing RNA; white interphase layer = containing DNA and protein; bottom layer = red organic phase containing DNA and protein). (3) RNA precipitation: Mix aqueous phase (top layer) with 0.5ml isopropanol per 0.7-0.75ml TriPure reagent (new eppe), Incubate 10min at RT, Spin 10min at 12,000rpm at 4°C, The RNA precipitate forms a gel-like pellet (often invisible before centrifugation) on the side and bottom of the tube. (4) RNA wash: Remove supernatant, Wash RNA pellet with at least 1ml 75% EtOH per 1ml TriPure reagent used for the initial RNA isolation step, Vortex, Spin 5min at 7,500xg at 4°C. (5) Redissolve the RNA: Air-dry RNA pellet (5-10min) (Do not let RNA pellet dry out completely as this will greatly decrease its solubility), Redisolve in RNAse free water, Pass solution a few times through a pipet tip and incubate 10min at 55°C-60°C to avoid problems with partially dissolved RNA, Expected yield for fibroblasts: 5-7µg from 1*106 cells. (6) Store RNA samples at -80°C (1µg needed for complete exp)
Label
Cy3
Label protocol
RNA concentration and purity were determined spectrophotometrically using the NanoDrop ND-1000 (NanoDrop Technologies), and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 100ng of total RNA spiked with 10 viral polyA transcript controls (Agilent) was converted to double-stranded cDNA in a reverse transcription reaction. Subsequently, the sample was converted to antisense cRNA, amplified and labeled with Cyanine 3-CTP (Cy3) in an in vitro transcription reaction according to the manufacturer's protocol (Agilent).
Hybridization protocol
A mixture of purified and labeled cRNA (Cy3 label: 825 ng; Cy5 label: 825 ng) was hybridised on Agilent's Mouse Whole Genome v2 oligo microarray (Cat # G2519F-026655, Agilent) followed by (manual) washing, according to the manufacturer's procedures.
Scan protocol
To assess the raw probe signal intensities, arrays were scanned using the Agilent DNA MicroArray Scanner with SuresScan High-Resolution Technology. Probe signals were quantified using Agilent's Feature Extraction software (version 10.7.3.1).
Description
MEF PSEN1&2-/- +hARF6 vs. MEF PSEN1&2-/- +hPSEN1 D257A/D385A
Data processing
We use the Agilent processed signal values (i.e., feature gProcessedSignal for the Cy3 signal and rProcessedSignal for the Cy5 signal) from Agilent Feature Extraction software v10.7.3.1 and compute log2-(Cy5/Cy3)-ratios. We remove Agilent control probes, as well as probes with signals below background on all arrays (absent spots). To decide whether a signal is significantly above background, we use the features gIsPosAndSignif and rIsPosAndSignif, also provided by the Agilent software. In case of multiple probes for the same Agilent ID, log2-(Cy5/Cy3)-ratios are averaged.
