S instructions. Quantitative RT-PCR was conducted in an ABI 7500 Real-Time PCR System (Applied Biosystem) using SYBR Green I (Invitrogen) to detect double-strand cDNA synthesis. Soybean F-BOX (F-Box protein family) and MET (insulin-degrading enzyme, metalloprotease) genes were used as reference genes for data normalization and to calculate the relative mRNA levels. Reactions were done in a volume of 20 L containing 10 L of cDNA, 0.1 ?SYBR Green I (Invitrogen), 0.025 mM dNTP, 1 ?PCR Buffer, 3 mM MgCl2, 0.25 U Platinum Taq DNA Polimerase (Invitrogen), and 200 nM of each reverse and forward primers. A negative control without cDNA template was included for each primer combination. Primer sequences for quantitative RT-PCR were as follows: for NFS1_Chr01, NFS1_01R 5′-CCTCCCAATTCTCTCCATCGGT-3′, and for NFS1_Chr11, NFS1_11R 5′-CCTCCCAATTTCCTCCATGGGC-3′ with the same forward PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27693494 primer NFS1F 5’CGGAGCACAAGTGCGTCC-3′; for NFS2_Chr09, NFS2_09R 5′-CCCGTGCACTTGAGCTGACA-3′, and for NFS2_Chr15, NFS2_15R 5′-Total proteins extract from untreated and cold stress treatment tissue were obtained from plants grown in the same conditions as described for quantitative RT-PCR. Protein quantification was performed by Bradford assay (BioRad) and equal amount (100 g) of protein was applied in a 7.5 native PAGE gel, AO and XDH were detected by activity staining previously described [29,53]Data analysisThreshold and baselines were manually determined using the ABI 7500 Real-Time PCR SDS Software v2.0. To analyze the relative cysteine desulfurases mRNA expression relative to the constitutive genes, we used the 2-Ct method [54]. Student’s t test was performed to compare pairwise differences in gene expression, following the two samples I-CBP112MedChemExpress I-CBP112 assuming unequal variances and two-tailed distribution parameters. For time-course treatment, 1-way ANOVA and Duncan post hoc analysis were performed using SPSS17. The means were considered significantly different when P < 0.05.Additional materialAdditional file 1: Alignment of IscS-like. Alignment of soybean cysteine desulfurase homologue to IscS from Escherichia coli. * indicates residues from active and from cofactor binding sites. # indicates amino acids residues that differ between soybean duplicated genes. Additional file 2: Alignment of SufS-like. Alignment of soybean cysteine desulfurase homologue to SufS from Escherichia coli. * indicates residues from active and from cofactor binding sites. # indicates amino acids residues that differ between soybean duplicated genes. Additional file 3: Alignment of ISD11 proteins. Alignment of soybean, Arabidopsis thaliana and Saccharomyces cerevisiae ISD11 proteins. Additional file 4: Primer sequences. Primer sequences and amplicon characteristics for each gene.Acknowledgements We are very grateful to Prof. Giancarlo Pasquali and Prof. M cia P. Margis from UFRGS for laboratorial and technical support. We also thank Prof. J gHeis et al. BMC Plant Biology 2011, 11:166 http://www.biomedcentral.com/1471-2229/11/Page 13 ofMeurer (Ludwig-Maximilians-University, Munich, Germany) for his editing of the manuscript. This work was supported by Conselho Nacional de Desenvolvimento Cient ico e Tecnol ico (CNPq- #302471/2009-0, #4703882009-9 and #473769/2007-7) of Brazil, and the Coordena o de Aperfei amento de Pessoal de Nivel Superior (PDEE/CAPES) of Brazil. Author details 1 Biotechnology Center, Federal University of Rio Grande do Sul - UFRGS, Porto Alegre, RS, Brazil. 2Department of Microbiology, Federal.
