We reverse transcribed 500 ng of high-quality DNase I-treated RNA samples into cDNA using oligo-dT primers and SuperScript™ III Reverse Transcriptase (Life Technologies). Subsequently, 1 µl of diluted cDNA was used for gene expression analyses with EVAGreen® qPCR Mix (Bio&Sell) and a C1000 thermocycler (Bio-Rad). Expression levels of biological triplicates were normalized to the reference genes ACT1 and GAPDH. Primers used for qPCR analyses are listed in Table S2 .
Qpcr mix evagreen
Manufactured by Bio&Sell
Sourced in Germany
QPCR Mix EvaGreen is a ready-to-use solution for quantitative real-time PCR (qPCR) analysis. It contains all the necessary components, including a DNA polymerase, buffer, and EvaGreen dye, which binds to double-stranded DNA and emits fluorescence during amplification.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid rt kit, by Thermo Fisher Scientific (2 mentions)
Sv total rna isolation system, by Promega (2 mentions)
Fastprep homogenizer, by MP Biomedicals (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
C1000 thermocycler, by Bio-Rad (1 mentions)
Cfx96 c1000 thermal cycler, by Bio-Rad (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Yeast dna extraction kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using qpcr mix evagreen
1
Quantitative Gene Expression Analysis
2
Quantifying Fungal Barcode Abundance
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Barcode-specific quantitative PCRs (qPCRs) were performed using DNA of fungal cells isolated from individual mice. Briefly, 100 ng of genomic DNA (gDNA) from plated inoculation pools or re-isolated strains was used to detect specific barcodes (Schwarzmüller et al., 2014 (link)) by using the EvaGreen QPCR Mix (Bio&Sell, Feucht, Germany) in a C1000/CFX96 Thermal Cycler (Bio-Rad, Munich, Germany). Barcode amounts were calculated by standard curves with the mutant's gDNA. At least three independent experiments were performed. Mutants with less than two measurable ct values were excluded.
3
Fungal Genomic DNA and RNA Isolation
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Fungal mycelium was lysed with glass beads by a FastPrep Homogenizer (MP Bio) for 2 min at 4.5 m s−1. Genomic DNA was isolated as described [21 (link)]. RNA was isolated with the SV Total RNA Isolation System (Promega) using the manufacturer′s protocol. RNA (1 µg) was treated with Baseline-ZERO DNase (Lucigen) and was reversely transcribed to cDNA by the RevertAid RT kit (Thermo) using anchored oligo-(dT)20 primers. Expression analysis was carried out with an AnalytikJena qTower3, using the qPCR Mix EvaGreen (Bio&SELL) and oligonucleotides with a minimum primer efficiency of 92 % (Table S1 ). Amplification procedure: initial denaturation, 95 °C, 15 min; 40 cycles of amplification (95 °C, 15 s; 60 °C, 20 s; 72 °C, 20 s). A melting curve was monitored by heating from 60 to 95 °C. The housekeeping reference genes, encoding α-actin (actB) and glycerinaldehyde-3-phosphate dehydrogenase (gpdA), served as internal standard. Gene expression levels were determined as described [25 (link)].
4
Quantitative PCR for C. albicans detection
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DNA was isolated from kidneys infected with either the WT (THE1-CIp10) or t-EED1 in the presence of doxycycline. Kidneys were homogenized and centrifuged for 15 min at 1500 × g. DNA was extracted from pellets using the Yeast DNA Extraction Kit (Thermo Scientific) following manufacturer’s instructions. For amplification of the C. albicans 18 S rRNA gene RDN18 the following primers were used: sense amplification primer, 5’-GGACCCAGCCGAGCCTT-3’ and antisense amplification primer, 5’-AAGTAAAAGTCCTGGTTCGCCA-3’30 (link). Quantitative PCR was conducted using 1 µl of template DNA and the QPCR Mix EvaGreen (Bio&SELL) on a CFX 96 Real time System (BioRad). The following condition were used for product amplification: 95 °C for 15 min, 40 cycles of each 95 °C for 15 s, 59 °C for 15 s and 72 °C for 15 s. To confirm PCR product specificity, a melting curve was generated. The resulting Ct values were plotted against the CFU determined from the homogenized tissue.
5
Fungal Transcriptome Profiling by qPCR
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Fungal mycelium from 36 h cultures in AMM, PDB and meat medium was lysed with glass beads (1–5 mm) in a FastPrep Homogenizer (MP Bio) for 2 min at 4.5 m s−1. Genomic DNA was isolated as described [38 (link)]. RNA was isolated with the SV Total RNA Isolation System (Promega) using the manufacturer’s protocol. RNA (1 µg) was DNase-treated (Baseline-ZERO, Lucigen) and was reversely transcribed into cDNA by the RevertAid RT kit (Thermo) using anchored oligo-(dT)20 oligonucelotides. Expression analysis was performed at the AnalytikJena qTower3 using the qPCR Mix EvaGreen (Bio&SELL) and oligonucleotides with a minimum primer efficiency of 95% (Additional file 45 : Table S6). After an initial denaturation at 95 °C for 15 min, 40 cycles of amplification were run (95 °C, 15 s; 60 °C, 20 s; 72 °C, 20 s). The housekeeping reference genes encoding actin (actA, DL89DRAFT_257372), the TEF transcription factor (tefA, DL89DRAFT_11075) and the glyceraldehyde-3-phosphate dehydrogenase (gpdA, DL89DRAFT_277503) served as internal standards. Gene expression levels were determined as described by Pfaffl [114 (link)].
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