C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 µl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated for 5 min (65°C) before subjected to 2x freeze-thawing. Lysates were clarified by centrifugation and the RNA precipitated with isopropyl alcohol/0.3 M sodium acetate by incubating for 1 h at -20°C. Precipitated pellets were washed (2x 1 ml 70% ice-cold ethanol), resuspended in DEPC-treated water and stored at -80°C. RNA integrity and concentration was confirmed using a Bioanalyzer (Agilent). RNA (500 ng) was treated with DNase (Epicenter) and cDNA synthesized using Reverse Transcriptase Superscript III (Invitrogen). cDNA samples were used for qPCR with EVAgreen mix (Bio&Sell). Primers (ACT1-F and ACT1-R for actin, ECE1-F and ECE1-R for ECE1 - Extended Data Table 4 ) were used at a final concentration of 500 nM. qPCR amplifications were performed using a Biorad CFX96 thermocycler. Data was evaluated using Bio-Rad CFX Manager 3.1 (Bio-Rad) with ACT1 as the reference gene and t0 as the control sample.
Evagreen mix
Manufactured by Bio&Sell
Sourced in Germany
EvaGreen mix is a solution designed for qPCR and PCR applications. It contains a fluorescent dye that binds to double-stranded DNA, allowing for real-time monitoring of the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Cfx96 thermocycler, by Bio-Rad (5 mentions)
Dnase, by Illumina (5 mentions)
Cfx manager 3, by Bio-Rad (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Rnapure, by Avantor (2 mentions)
Prism 5, by GraphPad (2 mentions)
High capacity rna to dna kit, by Thermo Fisher Scientific (2 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Prism version 6, by GraphPad (1 mentions)
7 protocols using evagreen mix
1
Analyzing Candida albicans gene expression
2
Analyzing Candida albicans gene expression
3
Quantitative PCR Validation of Transcriptome
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To confirm the results of transcriptome analysis, selected genes were subjected to qRT-PCR (for list of genes and primers see Supplemental Table S3). RNA samples were reverse transcribed into cDNA using the High Capacity RNA-to-DNA Kit (Applied Biosystems). cDNA was analyzed by real-time quantitative PCR (qPCR) using an ABI 7500 Fast Real Time PCR system (Applied Biosystems) and a pair of gene-specific primers for each selected gene. qPCR was performed in triplicates using EvaGreen Mix (Bio&SELL) and 20 ng of cDNA per reaction. The transcript levels of the examined genes were normalized to the geometric mean of four housekeeping genes (GAPDH, Dync1h1, Actb, Rpl41) (Thomas et al. 2014 (link)) from the same sample used as an internal reference and the fold change of mutant relative to the WT mice was calculated as 2−ΔΔCT (Pfaffl 2004 ). Statistical analysis was performed with GraphPad Prism 5.0 software, unpaired Student's t-test was used to estimate significance.
4
qRT-PCR Validation of Transcriptome Analysis
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To confirm the results of transcriptome analysis, selected genes were subjected to qRT-PCR (for list of genes and primers see Supplementary Table S4 ). RNA samples were reverse transcribed into cDNA using High Capacity RNA-to-DNA Kit (Applied Biosystems, Foster City, CA, USA). cDNA was analyzed by real-time quantitative PCR (qPCR) using an ABI 7500 Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA) and a pair of gene-specific primers for each selected gene. qPCR was performed in triplicates using EvaGreen Mix (Bio&SELL, Feucht, Germany) and 20 ng of cDNA per reaction. The transcript levels were normalized to the geometric mean of three housekeeping genes (Hmbs, Ywhaz, Actb) [35 (link),36 (link)] from the same sample used as an internal reference, and the fold change of mutant relative to the WT mice was calculated as 2−ΔΔCT [37 ]. Statistical analysis was performed with GraphPad Prism 5.0 software; unpaired Student’s t-test was used to estimate significance.
5
Quantitative Analysis of ECE1 Expression
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RNA (500 ng) was treated with DNase (Epicentre), and cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen). cDNA samples were used for qPCR with EvaGreen mix (Bio&Sell). Primers (ACT1-F and ACT1-R for ACT1 and ECE1-F2 and ECE1-R for ECE1 [Table S4 ]) were used at a final concentration of 500 nM. qPCR amplifications were performed using a CFX96 thermocycler (Bio-Rad). ECE1 expression was calculated using the threshold cycle (ΔΔCT) method, with ACT1 as the reference gene and C. albicans reference strain SC5314 (yeast morphology) as the control sample.
6
Quantitative Expression Analysis of ECE1 in Candida albicans
7
Quantitative Analysis of ECE1 Expression
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Total RNA was extracted from yeast-phase cells grown in YPD medium at 30°C or hyphae grown in RPMI 1640 according to the method described in reference 51 (link). RNA (500 ng) was treated with DNase (Epicentre), and cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen). cDNA samples were used for quantitative PCR with EvaGreen mix (Bio&SELL). Primers (ACT1-F/R and ECE1-F/R, Table S1 ) were used at a final concentration of 500 nM. qPCR amplifications were performed using a CFX96 thermocycler (Bio-Rad). ECE1 expression was calculated using the threshold cycle (ΔΔCT) method, with ACT1 as the reference gene and C. albicans reference strain SC5314 (yeast morphology) as the control sample.
Results of three biological replicates were analyzed in Prism version 6 (GraphPad, San Diego, CA).
Results of three biological replicates were analyzed in Prism version 6 (GraphPad, San Diego, CA).
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