Finally, SPIA cDNA was purified using QIAquick PCR Purification Kit and quantified using a Nanodrop ND 100 spectrophotometer. RT PCR Total RNA was extracted from Stage 8 whole embryos using NucleoSpin RNA II isolation Kit following the manufactures Ganetespib IC50 protocol. The quality and quantity of RNA were determined using Agilent RNA Nano LabChip. Approximately 300 ng of total RNA with a an RNA integrity number 8 were used for cDNA Inhibitors,Modulators,Libraries synthesis using ImProm II Reverse Transcription System and random primer hexamers according to the manufacturers instructions. For CM and RPE, the amplified SPIA cDNA was used as a template in the PCR reactions. All PCR reac tions were performed using PlatinumTaq DNA poly merase and the intron spanning primers are listed in Additional file 3, Table S1.
Amplification conditions included denaturation at 95 C for 1 min, annealing at 55 C for 2 min, and exten sion at 72 C for 30 s. All the RT PCR reactions were run from at least three independent biological samples and the fragments were gel purified Inhibitors,Modulators,Libraries and sequenced to confirm the specificity of the sequence. Quantitative RT PCR RT qPCR was performed using a 20 ul mixture containing 5 ul SPIA cDNA, 10 ul 2 SYBR Green Fluorescein qPCR Master Mix, and a 500 nM final concentration of the primers. Splice junction specific primers were designed using Primer 3 available in and were optimized following guidelines for RT qPCR experiments. Primers sequences and Ensembl or GenBank identification numbers are pro vided in Additional file 3, Table S2. Amplifications re actions were performed in triplicate using an iCycler.
The cycling conditions in cluded 10 min polymerase activation at 95 C and 35 cycles of 15 s at 95 C and 1 min at 60 C, followed by a dissoci ation run from 65 C to 95 C for melting curve analysis. The comparative Inhibitors,Modulators,Libraries cycle at threshold and an un paired Students t test analysis Inhibitors,Modulators,Libraries were used to determine relative changes in transcript levels compared to gapdh mRNA levels as previously reported using SigmaPlot 8. 0 Software. All analyses were performed in triplicate with at least three independent biological samples. Antibodies Antibodies against Sox 2, c Myc and Lin 28 were purchased from Santa Cruz Biotechnol ogy. Antibody against Klf4 was purchased from Aviva Systems Biology. Antibodies against Mitf, GFP and BrdU were pur chased from Abcam. Antibody against Pax 6 was obtained from the Developmental Studies Hybridoma Bank.
Anti Chx 10 antibody was purchased from ExAlpha. Antibody against p27Kip1 was obtained from BD Biosciences. All secondary Inhibitors,Modulators,Libraries antibodies were purchased from Molecular Probes and used at 1,100 dilution. Immunohistochemistry Embryos were fixed in 4% paraformaldehyde in PBS for 4 h at room temperature, equilibrated in 30% sucrose, embedded in selleck screening library OCT compound, and sec tioned at 12 um.