Global Gene Response in Saccharomyces Cerevisiae Exposed to Silver Nanoparticles

Supplementary Information

Global gene response in Saccharomyces cerevisiae exposed to silver nanoparticles

Javed H. Niazi1,2,3, Byoung-In Sang2, Yeon Seok Kim1, Man Bock Gu1

1College of Life Sciences and Biotechnology, Korea University, Sungbuk-gu, Seoul, Rep. of Korea

2Center for Environmental Technology Research, Korea Institute of Science and Technology, Sungbuk-gu, Seoul, Rep. of Korea

3Faculty of Engineering and Natural Sciences, Sabanci Univesity, Orhanli, 34956 Tuzla, Istanbul, Turkey

Materials and methods

Real time RT-PCR

The primers and probe sets used in this study are shown in Table S1. The reverse transcription (RT) reaction mixture contained 2.5 µg of total RNA, 1X TaqMan RT buffer, 5.5 mM magnesium chloride, 500 µM of each dNTP, 2.5 µM olig-dT, 0.4 U µL-1 RNase inhibitor, and 1.25 U µL-1 MultiScribe reverse transcriptase in a total reaction volume of 100 µL. Amplification for RT was done as follows: initial incubation step at 25 ºC for 10 min, followed by a reverse transcription step at 48 ºC for 30 min and a reverse transcriptase inactivation step at 95 ºC for 5 min in a GeneAmp® PCR system 9700 (Applied Biosystems, USA). The real-time PCR reactions were performed in a MicroAmp optical 96-well plate using the ABI PRISM 7000 sequence detection system (Applied Biosystems) with a 1X TaqMan Universal PCR Master mix, 2.5 µL of total cDNA from the RT reaction sample, and the primers and probe sets for either the test genes (CUP1-1, YHR138C, and BOP2)or ACT1 (endogenous gene) in a total reaction volume of 30 µL. Amplification was carried out using the following program: incubation step at 50 ºC for 2 min, followed by Taq DNA polymerase activation at 90 ºC for 10 min and 40 cycles of 95 ºC for 15 sec and 60 ºC for 1 min.

The threshold cycle (Ct) values were obtained and these values were used to calculate the expressions of COP1-1, YHR138C, and BOP2 based on the formula, 2ΔCt test gene (control-test)/2ΔCt endogenous gene (control-test), where, ΔCT of endogenous ACT1 gene (control-test) represents CT value of the endogenous ACT1 gene of the control (untreated) – CT value of the endogenous gene from the test sample (chemical treated). The ΔCT value for the target genes COP1-1/ YHR138C/ BOP2 (control-test) represents CT value for the target genes (COP1-1/ YHR138C/ BOP2) in the control (untreated) sample – the CT value of the target genes (COP1-1/ YHR138C/ BOP2) in the treated sample [1, 2].

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Table S1. Primers and probe sets used for confirmation of expression of CUP1-1, YHR138C, and BOP2 by real time RT-PCR. An housekeeping gene ACT1 was used as an endogenous control.

aReporter probes were labeled at the 5’ end with the fluorescent reporter dye 6-carboxy-fluorescein (5’-FAM) and the 3’ end with a non-fluorescent quencher (NFQ).

Table S2. Summary of commonly expressed genes in S. cerevisiae 288C exposed to AgNPs and Ag-ions.

Table S3. Summary of differentially expressed genes in S. cerevisiae 288C exposed to AgNPs (140 M) at 120 min and their expression levels after 210 min of exposure. The genes listed in table were found to be normally expressed to Ag-ions after 120 min of exposure.

*Up-regulated genes that showed ≥2-fold expression either at 120 or 210 min of AgNP exposure are only listed in the table. Those highlighted in bold indicates genes differentially responded.

Table S4. Summary of differentially expressed genes in S. cerevisiae 288C exposed to Ag-ions for 120 min and comparison with their expression folds to AgNPs exposure after 120 and 210 min.

*Only genes that showed ≥2 fold (Log2 ratio = ≥1) induction in at least one case are shown in the table.

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References

1.Livak K. J., Schmittgen T. D. (2001). Methods, 25(4):402-8.

2.Pfaffl M. W., Gerstmayer B., Bosio A., Windisch W. (2003). J. Nutr. Biochem., 14(12):691-702.

Figure S1. Effect of various concentrations of (A) silver nanoparticles (AgNPs) and (B) silver ions (Ag-ions) on growth of S. cerevisiae 288C in YPD broth.

Figure S2. Gene induction folds of selected three candidate genes (CUP1-1, YHR138C, and BOP2) chosen to validate their responses by RT-qPCR analysis. The primers and probe sets are shown in Table S1 of Supplementary Information. The induction folds of the representative genes were obtained from an independent experiment to ascertain their expression patterns correlate to the results found in microarray analysis. The expression ratio’s of treated samples were calculated relative of their untreated samples and ACT1 was used as an endogenous control gene. The expression ratio of CUP1-1, YHR138C, and BOP2 gene calculated as 2ΔCt target gene (control – test)/2ΔCt endogenous gene (control – test).

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