abcc.ncifcrf.gov/tools.jsp. RT- PCR validation cDNA amplified in vitro as described above was diluted 100-fold and 1 μl of this dilution PD-1/PD-L1 Inhibitor 3 was amplified by PCR. PCR was performed in 20-μl capillary tubes using a LightCycler (Roche Diagnostics, Indianapolis, Indiana) thermal cycler. Reaction mixtures contained 1× LC-Fast Start DNA master mix for SYBR Green I (Roche Diagnostics), 3 mM MgCl2, 20 pmol
each of forward and reverse primers, and 1 μl of cDNA template. The primer sequences are shown in Table 2. The PCR program included a denaturation step of 10 min at 95°C followed by 45 cycles of 1 s at 95°C, annealing for 8-9 s, and a 8-s extension at 72°C. Following amplification, the PCR products were subjected to melting curve analysis by raising the temperature from 45 to 95°C at a rate of 0.05°C/s. During the initial optimization phase PCR products were also electrophoresed on agarose gels to ensure that products of the correct size were amplified. Because trophozoites and cysts originated from selleck chemicals assemblage A and B, respectively, we verified that the PCR results were not affected by the genotype. Equivalent amounts of DNA from assemblage A isolate WB and assemblage B isolate GS were amplified in parallel using primers specific for portion of the ubiquitin, histone H2B and 14-3-3 protein shown in Table 2. No systematic bias that could be linked to the genotype was observed.
Disclaimer The comments and views detailed herein may not necessarily reflect the views of the WateReuse Research Foundation, 4SC-202 concentration BCKDHA its officers, directors, employees, affiliates or agents. Data deposition Microarray
data were deposited in the GEO database [GPL:11228]. Acknowledgements We gratefully acknowledge the WateReuse Research Foundation’s financial, technical, and administrative assistance in funding and managing the project through which this information was discovered. This project was funded in part by the National Institute of Allergy and Infectious Diseases (grant AI083719). Giardia lamblia microarrays and universal standard probe were obtained through NIAID’s Pathogen Functional Genomics Resource Center, managed and funded by the Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS and operated by the J. Craig Venter Institute. Our thanks to Phyllis Spatrick, UMass Worcester Genomics core facility, for help with microarray scanning and to the WateReuse Foundation Project Advisory Committee (Collin Balcombe, Walter Jakubowski, Paul Rochelle, Hal Stibbs, Shawn Thompson) for valuable advice and feedback. Electronic supplementary material Additional file 1: Comparison of Cy3 fluorescence emitted by microarrays hybridized with assemblage A and B trophozoite cDNA. Fluorescence values are means of two replicate microarray spots and are ranked in order of decreasing intensity, as in Figure 1. All datasets are biologically independent; the 3-digit microarray number is shown in the legend.