Embryonic stem cells derived from the FVB/N mouse strain
Treatment protocol
Cells were cultivated in the presence of of LIF
Extracted molecule
total RNA
Extraction protocol
RNA extraction with QIAGEN RNeasy Kit
Label
Biotin
Label protocol
cDNA synthesis 1st round of amplification. RNA was combined with 1ug of T7-dT primer (5’-GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG-(dT)24-3’) in a total volume of 12 ul and incubated at 70°C for 10 min. The reaction was placed on ice and the following reagents added in a total volume of 20 ul: first strand buffer (1x), DTT (10 mM), dNTP mix (500 uM), and Superscript III (200 units, Invitrogen) and incubated at 42°C for 2 h. Second strand synthesis was performed by adding the following reagents in a total volume of 150 ul: second strand buffer (1x), dNTP mix (200 uM), E. coli DNA ligase (10 units), E. coli DNA polymerase (40 units), RNase H (2 units), and incubated at 16°C for 2 h. T4 DNA polymerase (10 units) was then added to fill in the ends of the cDNA and incubated at 16°C for an additional 15 min. Following phenol:chloroform extraction and ethanol precipitation, the cDNA was resuspended in RNase-free water. Transcription was performed using Ambion’s (Austin, USA) MEGAscript reagents. A 20 ul reaction containing cDNA, NTP mix (7.5 mM), reaction buffer (1x), and 2 ul enzyme mix was incubated at 37°C for 4 h and the primary cRNA was purified using the RNeasy kit (Qiagen, Chatsworth, CA) per the manufacturer’s specifications. Two hundred nanograms of the purified cRNA were then carried forward in the secondary amplification. Samples with less than 200 ng cRNA were concentrated to a 5 ul volume and the entire sample used for the next round of amplification. cDNA synthesis 2nd round of amplification. First-strand cDNA was synthesized by incubating the above cRNA with 1 ug of random primers (Invitrogen, Carlsbad, CA) at 70°C for 10 min. The reaction was then placed on ice and the following reagents added in a 20-uL reaction: first-strand buffer (1x), DTT (10 mM), dNTP mix (500 uM), and Superscript III (200 units). The reaction was incubated at 42°C for 2 h. RNase H (2 units) was added to the reaction and incubated at 37°C for 20 min and then heat-inactivated. The cDNA was combined with 1 ug of the T7-dT primer and incubated at 70°C for 10 min. The reaction was then placed on ice and the following reagents added for a final volume of 150ul: second-strand buffer (1x), dNTP mix (200 uM), E. coli DNA Polymerase (40 units), and then incubated at 16°C for 2 h. T4 DNA polymerase (10 units) was added to fill in the ends of the cDNA and incubated at 16°C for an additional 15 min. The cDNA was purified with phenol:chloroform and prec ipitated with ethanol. Biotin secondary IVT. The secondary transcription reaction was performed using the Enzo BioArray HighYield RNA kit (Affymetrix, Santa Clara, CA). The secondary cDNA was incubated at 37°C for 4 h in a 40 ul reaction as indicated by in the manufacturer recommendations and the labelled cRNA purified using the RNeasy kit.
Hybridization protocol
Prior to injection of hybridization cocktail, probes were primed by injection with hybridisation buffer (100mM MES, 1M [Na+], 20mM EDTA, 0.01% Tween-20 and placed in oven (45oC) for 10 minutes with rotation (60rpm). Hybridisation was performed by injection of the hybridization cocktail and incubating the probe in oven at 45oC with rotation (60rpm) for 16 hours. Probes were washed at the Fluidics FS-450 station with Buffer A (6X SSPE, 0.01% Tween 20) and B (100mM MES, 0.1M [Na+], 0.01% Tween-20). Staining of the probes was performed with 0.01mg/ml SAPE (Streptavidin Phycoerythrin) and biotinylated antibody (0.06mg/ml).
