Quantitative Real-Time PCR Analysis of Fungal Transcripts

Quantitative PCR (qPCR) was performed in a Rotor-Gene Q system (Qiagen, Hilden, Germany). Reactions were performed in technical duplicates or triplicates. The amplification mixture (final volume 15 μL) contained 7.5 μL 2 × iQ SYBR Green Mix (Bio-Rad, Hercules, California, USA), 100 nM forward and reverse primer, and 2.5 μL cDNA (diluted 1:20). Primer sequences and cycling conditions are provided in Table 2. Data normalization using sar1 and act as reference genes and calculations were performed as previously published [35 (link)].Table 2

Primer used for qPCR

Primer name	Sequence 5′–3′	References
actfw	TGAGAGCGGTGGTATCCACG	[35 (link)]
actrev	GGTACCACCAGACATGACAATGTTG	[35 (link)]
sar1fw	TGGATCGTCAACTGGTTCTACGA	[35 (link)]
sar1rev	GCATGTGTAGCAACGTGGTCTTT	[35 (link)]
xyr1f	CCCATTCGGCGGAGGATCAG	[35 (link)]
xyr1r	CGAATTCTATACAATGGGCACATGGG	[35 (link)]
taqxyn1 f	CAGCTATTCGCCTTCCAACAC	[13 (link)]
taqxyn1 r	CCAAAGTTGATGGGAGCAGAA	[13 (link)]
cbh1f	GATGATGACTACGCCAACATGCTG	[12 (link)]
cbh1r	ACGGCACCGGGTGTGG	[12 (link)]
cre1_a_f	ACCTCCTGAATCCAACGTCGG	[17 (link)]
cre1_a_r	TGGGTGCGAATGTGCCTGG	[17 (link)]
bga1f	CGTTTGATCCTTTCGGCGGCT	[19 (link)]
bga1r	CCAAAGGTCATGTATATGTTGAAGATGGTC	[19 (link)]
gal1f	GGAGGCATGGACCAGGC	[19 (link)]
gal1r	GACATGCTTGTTGGAGGTGACG	[19 (link)]
xorf	CTGTGACTATGGCAACGAAAAGGAG	[19 (link)]
xorr	CACAGCTTGGACACGTGAAGAG	[19 (link)]
st RT 1	CCGTCTACCGTCTGTTGTGC	[5 (link)]
st RT 2	GAAGTAGGAAAGAACCGCATTG	[5 (link)]

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Rassinger A., Gacek-Matthews A., Strauss J., Mach R.L, & Mach-Aigner A.R. (2018). Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator. Fungal Biology and Biotechnology, 5, 15.

Publication 2018

Rotor-Gene Q system 2 × iQ SYBR Green Mix

Cdna Gene system Genes Primer Sybr green

Corresponding Organization :

Other organizations : TU Wien, BOKU University

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Variable analysis

independent variables

Primer sequences

dependent variables

Gene expression levels

control variables

CDNA amount (2.5 μL, diluted 1:20)
Primer concentration (100 nM)
Reaction volume (15 μL)
Reaction components (7.5 μL 2 × iQ SYBR Green Mix)
Reference genes (sar1 and act) for data normalization

controls

Not explicitly mentioned
Not explicitly mentioned

Annotations

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