C. albicans (ATCC 10,231) was cultured in duplicate in 100 mL flasks at 30 °C with vigorous shaking in the presence of 4-AN at the concentration of MIC/4 (1 µg/mL) or 0.1% DMSO (control cells in duplicate). The cultures were terminated by centrifugation when OD600 reached a value of 1. Cells were frozen at −70 °C or used immediately. Total RNA was isolated from four cell samples using a YeaStarTM RNA kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol.
Yeastar rna kit
Manufactured by Zymo Research
Sourced in United States
The YeaStar RNA kit is a laboratory equipment product designed for the extraction and purification of RNA from yeast samples. It provides a reliable and efficient method for isolating high-quality RNA for downstream applications such as gene expression analysis, reverse transcription, and other molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by New England Biolabs (4 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Smart first strand cdna synthesis kit, by EURx (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Iscript reverse transcription supermix for rt qpcr, by Bio-Rad (2 mentions)
Oligo dt, by Thermo Fisher Scientific (2 mentions)
Rna cleanup kit, by New England Biolabs (2 mentions)
Faststart essential dna green master, by Roche (2 mentions)
Lightcycler, by Roche (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
K2po4, by Merck Group (1 mentions)
Probe qpcr master mix, by EURx (1 mentions)
Rotor gene 6000, by Qiagen (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
27 protocols using yeastar rna kit
1
RNA Isolation from C. albicans under Antifungal Stress
2
Transcriptional Analysis of C. albicans
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Total RNA was isolated from exponentially growing C. albicans cells using the YeaStarTM RNA Kit (Zymo Research, Irvine, U.S.A) according to the manufacturer's instructions. The RNA was treated with DNase I (Invitrogen, Paisley, UK) in the presence of RNase inhibitor (Rnase OUT: Invitrogen), and then the levels of specific transcripts subjected to qRT‐PCR using published procedures (Leach et al., 2012a ). The primers are described in Supporting Information Table S2.
3
Silymarin Regulates SAP4 Expression in C. albicans
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The expression of the SAP4 gene was evaluated using real-time PCR after treatment with silymarin. The cells of C. albicans ATCC 10231 were exposed to silymarin at the concentration of 15 µg/mL (MIC/2) or 1% DMSO (control) during propagation in liquid culture in Spider medium (1% nutrient broth, 1% mannitol, 0.2% K2PO4 (Sigma-Aldrich), pH 7.2) with 10% fetal bovine serum (FBS) (Sigma-Aldrich). After 20-h incubation at 37 °C, the yeasts were collected and total RNA was extracted with the YeaStar RNA kit (Zymo Research, USA), according to the manufacturer’s instructions. cDNA was then synthesized using a Smart First Strand cDNA Synthesis Kit (EurX, Poland) following the manufacturer’s instructions. For PCR detection of transcripts, we used TaqMan gene expression assays (Lot: 170255, designed by manufacturer, Thermo Fisher Scientific, England) and the Probe qPCR Master Mix (EurX, Poland). The cDNA samples were pre-treated at 50 °C for 2 min with uracil-N-glycosylase to degrade any dUMP-containing PCR products and then subjected to initial denaturation at 95 °C for 10 min., followed by 40 amplification cycles with denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s using RotorGene-6000 (Corbett). The relative level of expression of the tested gene was calculated with the 2-(ΔΔCt) method using ACT1 as a reference gene [38 (link)].
4
Differential Expression of Candida Genes
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The Candida cells (ATCC 24433) were treated with CNMA as well as ML-CNMA. The cells (that include positive control/treated and negative control/non-treated) were pelleted and RNA was extracted by using RNA extraction kit (YeaStar RNA Kit, Zymo Research). Pure RNA was quantified and used to synthesize first strand cDNA as manufacturer’s instruction (cDNA synthesis kit, Fermentas, United States). PCR reactions were performed by using the primers HWP1 F 5′-CCA CTA CTA CTG AAG CCA AAT C-3′; HWP1 R 5′-AAG TGG ATA CTG TAC CAG TTG G-3′; EFB1 F 5′-AGT CAT TGA ACG AAT TCT TGG CTG-3′ and EFB1 R 5′-TTC TTC AAC AGC AGC TTG TAA GTC-3′. The following conditions were used for amplification of HWP1: 94°C for 5 min, 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, and 72°C for 30 s for 30 cycles and for housekeeping gene; primer EFB1: 94°C for 30 s, 60°C for 30 s, and 72°C for 60 s (21 cycles), respectively (Davis-Hanna et al., 2008 (link)).
5
Yeast Total RNA Extraction and RT-PCR
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Total RNA was isolated from yeast strains using YeaStar™ RNA Kit (Zymo Research).
Approximately 300 ng of total RNA was treated with DNase (Invitrogen) and used as template for cDNA synthesis by SuperScript III Reverse Transcriptase (Invitrogen). PCR was undertaken using Taq 2X Master Mix (New England Biolabs Inc.) using conditions as provided by the manufacturer. The RT-PCR of apyrase transcripts was undertaken with attB1 and attB2 primers (Table S1). The yeast UBC6 gene (ubiquitin-conjugating enzyme) was used as a control.
Approximately 300 ng of total RNA was treated with DNase (Invitrogen) and used as template for cDNA synthesis by SuperScript III Reverse Transcriptase (Invitrogen). PCR was undertaken using Taq 2X Master Mix (New England Biolabs Inc.) using conditions as provided by the manufacturer. The RT-PCR of apyrase transcripts was undertaken with attB1 and attB2 primers (Table S1). The yeast UBC6 gene (ubiquitin-conjugating enzyme) was used as a control.
6
Nab2 Overexpression Effects on mRNA Polyadenylation
7
Quinalizarin Modulates Candida Gene Expression
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The expression of HWP1, SAP4, ALS1, ALS3, HYR1, BCR1, EFG1, ECE1, and CPH1 genes was evaluated using real-time PCR after treatment with quinalizarin. The C. albicans cells were exposed to anthraquinone at the concentration of MIC/2 (4 µg/mL) or 1% DMSO (control) during propagation in liquid culture in Spider medium with 10% fetal bovine serum. After 10-h incubation at 37°C, the yeast cells were collected, and total RNA was extracted with the YeaStar RNA kit (Zymo Research, USA) according to the manufacturer’s instructions. cDNA was then synthesized using a Smart First Strand cDNA Synthesis Kit (EurX, Poland) following the manufacturer’s instructions. For PCR detection of transcripts, we used TaqMan gene expression assays (Lot: 170255, designed by the manufacturer, ThermoFisher Scientific, England) and the Fast Probe qPCR Master Mix (EurX, Poland). The cDNA samples were pre-treated with uracil-N-glycosylase at 37°C for 2 min to degrade any dUMP-containing PCR products and then subjected to initial denaturation at 95°C for 3 min, followed by 40 amplification cycles with denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s using QuantStudio3 (Applied Biosystems). The relative level of expression of the analyzed genes was calculated with the 2-(∆∆Ct) method using ACT1 as a reference gene (43 (link)).
8
Relative Quantification of CDR1 in C. albicans
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C. albicans strains were grown overnight in YPD medium at 30°C with shaking. Total RNA was extracted using a YeaStar RNA Kit (ZymoResearch, United States). Reverse transcription of the isolated RNA samples was performed by using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio, Japan). The cDNA abundance was relatively quantified using TB Green® Premix DimerEraser™ (Takara Bio, Japan) in a CFX96™ Real-Time PCR Detection System (Bio-Rad, United States) with the following strategy: 1) 95°C for 30 s; 2) 95°C for 5 s, 50°C for 30 s, and 72°C for 30 s, for 40 cycles. The relative expression level of the CDR1 gene was normalized to that of the reference ACT1 gene, and the data were interpreted as fold changes based on the untreated control according to the 2−ΔΔCt method and triplicate measurements were conducted with each sample (Lu et al., 2015 (link)).
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C. albicans strains were grown overnight in YPD medium at 30°C with shaking. Total RNA was extracted using a YeaStar RNA Kit (ZymoResearch, United States). Reverse transcription of the isolated RNA samples was performed by using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio, Japan). The cDNA abundance was relatively quantified using TB Green® Premix DimerEraser™ (Takara Bio, Japan) in a CFX96™ Real-Time PCR Detection System (Bio-Rad, United States) with the following strategy: 1) 95°C for 30 s; 2) 95°C for 5 s, 50°C for 30 s, and 72°C for 30 s, for 40 cycles. The relative expression level of the CDR1 gene was normalized to that of the reference ACT1 gene, and the data were interpreted as fold changes based on the untreated control according to the 2−ΔΔCt method and triplicate measurements were conducted with each sample (Lu et al., 2015 (link)).
9
Quantitative Gene Expression Analysis
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Total RNA was extracted using the YeaStar™ RNA Kit (Zymo Research). RNA was DNAse treated (DNAse I, NewEngland Biolabs) for 30 min. and subsequently purified using the RNA Clean & Concentrator™-5 Kit (Zymo Research). RNA was used for the reverses transcription reaction (iScript™ Reverse Transcription Supermix for RT-qPCR, Bio-Rad) and cDNA was used for SYBR Green qPCR (SsoAdvanced™ Universal SYBR® Green Supermix. Bio-Rad) using the Bio-Rad CFX Connect™. Primers used for the qPCR reaction are listed in Additional file 1 : Table S4. For Figs. 4 a and 5 a, b (top panel) total copy number was calculated from a standard curve with GAPDH as an internal standard. Fold changes in Fig. 5 a, b (bottom panel) were calculated under consideration of the reaction efficiency as previously described [59 (link)]. Log and stationary phase expression were compared with lag phase expression using transcript level normalized to GAPDH.
10
rRNA Depletion and RNA Sequencing
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RNA was isolated using the YeaStar RNA Kit (Zymo Research). Between step 5 and 6 of the protocol, the sample was treated with DNase I (Sigma-Aldrich). An in-column DNase digestion was performed according to Appendix A of RNA Clean & Concentrator-5 (Zymo Research). From the isolated RNA, rRNA was removed using the Ribo-Zero rRNA Removal Kit (Illumina). Samples were barcoded and prepared for sequencing using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs). All samples were pooled and sequenced using a NextSeq 500 sequencer (Illumina).
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