Act1 out of Action: Identifying Reliable Reference Genes in Trichoderma reesei for Gene Expression Analysis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fungal Strains
2.2. Transcriptome Dataset Processing and Analysis
2.3. Cultivation Conditions
2.4. RNA Extraction
2.5. cDNA Synthesis
2.6. qPCR
2.7. Data Analysis
3. Results
3.1. Identification of New Candidate Reference Genes for T. reesei Using WTS Data
3.2. Evaluation of Gene Expression Stability Using RT-qPCR and RefFinder
3.3. Use of bzp1 and tpc1 as Reference Genes for Analyzing Gene Expression Using RT-qPCR
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
DTT | Dithiothreitol |
RT-qPCR | Reverse transcription–quantitative polymerase chain reaction |
MA | Mandels–Andreotti |
DMF | Dimethylformamide |
SEB | Sugarcane exploded bagasse |
MM | Minimal medium |
CV | Coefficient of variation |
MIQE | Minimum information for publication of quantitative real-time PCR experiments |
WTS | Whole transcriptome sequencing |
Ct | Threshold cycle |
SRA | Sequence Read Archive |
BCM | Basic culture medium |
Appendix A
Appendix A.1
Run | SRA 1 Study | Sequenced Bases | File Size | Platform | Condition | Reference |
---|---|---|---|---|---|---|
SRR23952272 | SRP429031 | 7.6 G | 2.1 Gb | Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) | glucose R1 2 | [67] |
SRR23952273 | SRP429031 | 6.8 G | 1.9 Gb | Illumina NovaSeq 6000 | glucose R2 | |
SRR8698740 | SRP187914 | 7 G | 2.9 Gb | Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) | 2% lactose (transfer after 12 h) R1 | [68] |
SRR8698741 | SRP187914 | 7.8 G | 3.2 Gb | Illumina HiSeq 2000 | 2% lactose (transfer after 12 h) R2 | |
SRR5765024 | SRP110683 | 2.9 G | 2.0 Gb | Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) | 1% sugarcane bagasse 48 h R1 | [69] |
SRR5765025 | SRP110683 | 3.0 G | 2.0 Gb | Illumina HiSeq 2500 | 1% sugarcane bagasse 48 h R2 | |
SRR5765030 | SRP110683 | 3.2 G | 2.1 Gb | Illumina HiSeq 2500 | 1% sugarcane bagasse (transfer from 1% glycerol) R1 | |
SRR5765031 | SRP110683 | 4.0 G | 2.7 Gb | Illumina HiSeq 2500 | 1% sugarcane bagasse (transfer from 1% glycerol) R2 | |
SRR7761289 | SRP159003 | 2.8 G | 1.8 Gb | Illumina HiSeq 2000 | MA with 1% cellulose in constant light R1 | [70] |
SRR7761290 | SRP159003 | 2.7 G | 1.7 Gb | Illumina HiSeq 2000 | MA with 1% cellulose in constant light R2 | |
SRR7761293 | SRP159003 | 2.7 G | 1.7 Gb | Illumina HiSeq 2000 | MA with 1% cellulose in constant dark R1 | |
SRR7761294 | SRP159003 | 2.6 G | 1.6 Gb | Illumina HiSeq 2000 | MA with 1% cellulose in constant dark R2 | |
SRR8329346 | SRP173612 | 7.5 G | 2.7 Gb | Illumina HiSeq X Ten (Illumina, San Diego, CA, USA) | MM with 1% Avicell 48 h R1 | [71] |
SRR8329347 | SRP173612 | 6.9 G | 2.4 Gb | Illumina HiSeq X Ten | MM with 1% Avicell 48 h R2 | |
SRR8329344 | SRP173612 | 7.6 G | 2.7 Gb | Illumina HiSeq X Ten | MM with 1% Avicell + 1% DMF 48 h R1 | |
SRR8329345 | SRP173612 | 7.1 G | 2.5 Gb | Illumina HiSeq X Ten | MM with 1% Avicell + 1% DMF 48 h R2 | |
SRR8756161 | SRP188940 | 882.3 M | 368.0 Mb | Illumina NextSeq 500 (Illumina, San Diego, CA, USA) | 24 h on MA with glucose (transfer for 3 h to 1% D-Mannitol) R1 | [72] |
SRR8756162 | SRP188940 | 738.7 M | 306.6 Mb | Illumina NextSeq 500 | 24 h on MA with glucose (transfer for 3 h to 1% D-Mannitol) R2 | |
SRR25252694 | SRP448956 | 2.7 G | 908.6 Mb | Illumina NovaSeq 6000 | MA with 1% corn stover (4 h transfer) R1 | [73] |
SRR25252695 | SRP448955 | 2.7 G | 908.0 Mb | Illumina NovaSeq 6000 | MA with 1% corn stover (4 h transfer) R2 | |
SRR19551435 | SRP378722 | 5.9 G | 1.7 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-glucuronic acid (4 h transfer) R1 | |
SRR19551437 | SRP378720 | 4.5 G | 1.3 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-glucuronic acid (4 h transfer) R2 | |
SRR19551421 | SRP378737 | 6.7 G | 2.0 Gb | Illumina NovaSeq 6000 | MA with 25 mM L-arabinose (4 h transfer) R1 | |
SRR19551434 | SRP378723 | 6.9 G | 2.1 Gb | Illumina NovaSeq 6000 | MA with 25 mM L-arabinose (4 h transfer) R2 | |
SRR19551432 | SRP378725 | 5.3 G | 1.6 Gb | Illumina NovaSeq 6000 | MA with 25 mM L-rhamnose (4 h transfer) R1 | |
SRR19551433 | SRP378724 | 4.9 G | 1.5 Gb | Illumina NovaSeq 6000 | MA with 25 mM L-rhamnose (4 h transfer) R2 | |
SRR19551429 | SRP378728 | 6.8 G | 2.0 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-galacturonic acid (4 h transfer) R1 | |
SRR19551431 | SRP378726 | 6.9 G | 2.0 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-galacturonic acid (4 h transfer) R2 | |
SRR19551420 | SRP378736 | 6.1 G | 1.8 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-xylose (4 h transfer) R1 | |
SRR19551428 | SRP378729 | 7.4 G | 2.2 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-xylose (4 h transfer) R2 | |
SRR19551424 | SRP378733 | 5.1 G | 1.5 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-mannose (4 h transfer) R1 | |
SRR19551425 | SRP378732 | 3.9 G | 1.1 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-mannose (4 h transfer) R2 | |
SRR19551413 | SRP378744 | 4.5 G | 1.2 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-galactose (4 h transfer) R1 | |
SRR19551423 | SRP378734 | 4.8 G | 1.4 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-galactose (4 h transfer) R2 | |
SRR19551416 | SRP378741 | 4.2 G | 1.2 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-fructose (4 h transfer) R1 | |
SRR19551418 | SRP378739 | 4.2 G | 1.2 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-fructose (4 h transfer) R2 | |
SRR19551414 | SRP378743 | 5.2 G | 1.5 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-glucose (4 h transfer) R1 | |
SRR19551415 | SRP378742 | 6.6 G | 1.8 Gb | Illumina NovaSeq 6000 | MA with 25 mM D-glucose (4 h transfer) R2 |
Run | SRA 1 Study | Sequenced Bases | File Size | Platform | Condition | Reference |
---|---|---|---|---|---|---|
SRR24768099 | SRP440257 | 6.3 G | 2.0 Gb | Illumina NextSeq 2000 (Illumina, San Diego, CA, USA) | Fed batch with lactose + 10 mM DTT for 2 h R1 2 | [74] |
SRR24768104 | SRP440257 | 5.9 G | 1.9 Gb | Illumina NextSeq 2000 | Fed batch with lactose + 10 mM DTT for 2 h R2 | |
SRR24768100 | SRP440257 | 4.6 G | 1.4 Gb | Illumina NextSeq 2000 | Fed batch with lactose R1 | |
SRR24768103 | SRP440257 | 5.2 G | 1.6 Gb | Illumina NextSeq 2000 | Fed batch with lactose R2 | |
SRR24768101 | SRP440257 | 5.3 G | 1.6 Gb | Illumina NextSeq 2000 | Fed batch with glucose + 10 mM DTT for 2 h R1 | |
SRR24768102 | SRP440257 | 5.7 G | 1.7 Gb | Illumina NextSeq 2000 | Fed batch with glucose + 10 mM DTT for 2 h R2 | |
SRR24768105 | SRP440257 | 4.7 G | 1.4 Gb | Illumina NextSeq 2000 | Fed batch with glucose R1 | |
SRR24768106 | SRP440257 | 4.5 G | 1.3 Gb | Illumina NextSeq 2000 | Fed batch with glucose R2 | |
SRR4446960 | SRP091982 | 2.4 G | 1.6 Gb | Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) | BCM with 1% fructose 48 h + 24 h R1 | [75] |
SRR4446961 | SRP091982 | 2.5 G | 1.7 Gb | Illumina HiSeq 2000 | BCM with 1% fructose 48 h + 24 h R2 | |
SRR4446958 | SRP091982 | 2.5 G | 1.7 Gb | Illumina HiSeq 2000 | BCM with 1% fructose 48 h (transfer to 0.5% SEB 24) R1 | |
SRR4446959 | SRP091982 | 2.5 G | 1.7 Gb | Illumina HiSeq 2000 | BCM with 1% fructose 48 h (transfer to 0.5% SEB 24 h) R2 | |
SRR4446955 | SRP091982 | 2.3 G | 1.6 Gb | Illumina HiSeq 2000 | BCM with 1% fructose 48 h (transfer to 0.5% SEB 12 h) R1 | |
SRR4446956 | SRP091982 | 2.7 G | 1.8 Gb | Illumina HiSeq 2000 | BCM with 1% fructose 48 h (transfer to 0.5% SEB 12 h) R2 | |
SRR4446953 | SRP091982 | 2.5 G | 1.7 Gb | Illumina HiSeq 2000 | BCM with 1% fructose 48 h (transfer to 0.5% SEB 6 h) R1 | |
SRR4446954 | SRP091982 | 2.4 G | 1.7 Gb | Illumina HiSeq 2000 | BCM with 1% fructose 48 h (transfer to 0.5% SEB 6 h) R2 | |
SRR23088649 | SRP417642 | 6.6 G | 1.9 Gb | Illumina NovaSeq 6000 | 1% Avicell 48 h R1 | [76] |
SRR23088650 | SRP417642 | 7.2 G | 2.1 Gb | Illumina NovaSeq 6000 | 1% Avicell 48 h R2 | |
SRR23088646 | SRP417642 | 8.3 G | 2.4 Gb | Illumina NovaSeq 6000 | 1% Avicell 48 h + 3 mM Zn2+ R1 | |
SRR23088647 | SRP417642 | 7.1 G | 2.0 Gb | Illumina NovaSeq 6000 | 1% Avicell 48 h + 3 mM Zn2+ R2 | |
SRR28595945 | SRP500486 | 7.9 G | 2.4 Gb | Illumina HiSeq 4000 (Illumina, San Diego, CA, USA) | high-melanin necromass from Hyaloscypha bicolor R1 | [77] |
SRR28595946 | SRP500486 | 8.5 G | 2.6 Gb | Illumina HiSeq 4000 | high-melanin necromass from H. bicolor R2 | |
SRR28595948 | SRP500486 | 8.0 G | 2.5 Gb | Illumina HiSeq 4000 | low-melanin necromass from H. bicolor R1 | |
SRR28595949 | SRP500486 | 8.5 G | 2.6 Gb | Illumina HiSeq 4000 | low-melanin necromass from H. bicolor R2 |
Appendix B
Appendix B.1
- Indexing of genomes for further use in HISAT2:hisat2-build genome.fa genome
- Writing a SAM file from paired-end RNA-seq files:hisat2 -x genome -1 sample_R1.fastq.gz -2 sample_R2.fastq.gz -S output.sam
- Writing a SAM file from single-end RNA-seq file:hisat2 -x genome_index -U sample.fastq.gz -S output.sam
- Conversion from SAM to BAM format via SAMtools:samtools sort -l 9 -o output.bam -@ 4 output.sam
- Extraction of raw counts from the SAM file:featureCounts -p -t exon -g gene_id -a annotation.gtf -o output.txtoutput.bam
- If (!requireNamespace(“BiocManager”, quietly = TRUE)) {install.packages(“BiocManager”)}BiocManager::install(“DESeq2”)BiocManager::install(“EnhancedVolcano”)install.packages(“writexl”)library(writexl)library(DESeq2)library(EnhancedVolcano)
- cond1_1 <- read.table(“C:/---/cond1_1.txt”, header = TRUE, sep =“\t”, stringsAsFactors = FALSE)cond1_2 <- read.table(“C:/---/cond1_2.txt”, header = TRUE, sep =“\t”, stringsAsFactors = FALSE)cond2_1 <- read.table(“C:/---/cond2_1.txt”, header = TRUE, sep =“\t”, stringsAsFactors = FALSE)cond2_2 <- read.table(“C:/---/cond2_2.txt”, header = TRUE, sep =“\t”, stringsAsFactors = FALSE)
- countData1_cond1_1 <- cond1_1[, 7:ncol(cond1_2)]countData1_cond1_2 <- cond1_2[, 7:ncol(cond1_2)]countData2_cond2_1 <- cond2_1[, 7:ncol(cond2_1)]countData2_cond2_2 <- cond2_1[, 7:ncol(cond2_2)]
- countData1_cond1_1 <- as.data.frame(countData1_cond1_1)countData1_cond1_2 <- as.data.frame(countData1_cond1_2)countData2_cond2_1 <- as.data.frame(countData2_cond2_1)countData2_cond2_2 <- as.data.frame(countData2_cond2_2)
- rownames(countData1_cond1_1) <- cond1_1$Geneidrownames(countData1_cond1_2) <- cond1_2$Geneidrownames(countData2_cond2_1) <- cond2_1$Geneidrownames(countData2_cond2_2) <- cond2_2$Geneid
- countData<-cbind(countData1_cond1_1,countData1_cond1_2,countData2_cond2_1, countData2_cond2_2)colnames(countData) <- c(“Condition1 1”, “Condition1 2”)
- ncol(countData)
- sampleInfo <- data.frame(row.names = colnames(countData),condition = factor(rep(c(“Condition1”, “Condition2”), each = 2)),replicate = factor(rep(c(“Rep1”, “Rep2”), 2)))
- dds <- DESeqDataSetFromMatrix(countData = countData, colData =sampleInfo, design = ~ condition)dds <- estimateSizeFactors(dds)normalized_counts <- counts(dds, normalized=TRUE)normalized_counts_df <- as.data.frame(normalized_counts)normalized_counts_df$GeneID <- rownames(normalized_counts_df)normalized_counts_df <- normalized_counts_df[,c(ncol(normalized_counts_df), 1:(ncol(normalized_counts_df– - 1))]
- write_xlsx(normalized_counts_df“ “C:/---/normalized_counts.xl”x”)
Appendix B.2
Strain | C-Source | Time Point | Replicate | Ct Values | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|
sar1 | act1 | bzp1 | tpc1 | cue1 | ubi1 | sas3 | cbh1 | ||||
QM6a | Cellulose | 120 | 1 | 19.80 | 19.56 | 18.50 | 19.55 | 18.83 | 19.48 | 20.59 | 17.00 |
QM6a | Cellulose | 120 | 2 | 21.45 | 21.80 | 18.51 | 19.60 | 19.83 | 19.81 | 21.29 | 18.45 |
QM6a | Cellulose | 48 | 1 | 18.46 | 17.35 | 19.26 | 20.74 | 19.57 | 20.59 | 22.09 | 11.00 |
QM6a | Cellulose | 48 | 2 | 18.78 | 18.04 | 18.59 | 19.79 | 18.91 | 19.83 | 21.01 | 11.48 |
QM6a | DTT | 6 | 1 | 19.84 | 22.19 | 19.97 | 22.40 | 19.60 | 21.08 | 21.66 | 24.65 |
QM6a | DTT | 6 | 2 | 20.51 | 21.92 | 19.60 | 21.75 | 19.69 | 19.97 | 21.04 | 24.55 |
QM6a | DTT | 48 | 1 | 21.38 | 23.29 | 18.35 | 19.05 | 18.97 | 18.51 | 19.37 | 26.00 |
QM6a | DTT | 48 | 2 | 21.90 | 24.03 | 18.51 | 19.34 | 19.66 | 18.63 | 19.77 | 26.55 |
QM6a | Glucose | 36 | 1 | 23.96 | 22.87 | 17.18 | 18.04 | 17.38 | 17.36 | 18.40 | 30.50 |
QM6a | Glucose | 36 | 2 | 23.95 | 22.68 | 17.27 | 17.97 | 17.45 | 17.42 | 18.45 | 30.60 |
QM6a | Glucose | 84 | 1 | 23.44 | 22.54 | 18.41 | 19.24 | 18.89 | 19.10 | 20.35 | 30.65 |
QM6a | Glucose | 84 | 2 | 24.87 | 24.10 | 18.58 | 19.61 | 19.75 | 19.62 | 21.09 | 31.00 |
QM6a | Glycerin | 84 | 1 | 23.83 | 24.88 | 21.37 | 21.98 | 22.19 | 20.77 | 22.01 | n.m. 1 |
QM6a | Glycerin | 84 | 2 | 24.10 | 25.60 | 20.07 | 20.96 | 21.28 | 19.59 | 21.09 | n.m. |
QM6a | Glycerin | 24 | 1 | 17.08 | 16.81 | 18.72 | 19.52 | 17.93 | 19.26 | 20.89 | n.m. |
QM6a | Glycerin | 24 | 2 | 17.26 | 16.08 | 19.02 | 19.69 | 17.97 | 19.26 | 20.87 | n.m. |
QM6a | Lactose | 96 | 2 | 20.81 | 20.13 | 19.03 | 20.06 | 19.99 | 18.63 | 19.68 | 19.70 |
QM6a | Lactose | 96 | 1 | 21.55 | 20.33 | 17.26 | 18.30 | 18.19 | 16.47 | 17.73 | 24.05 |
QM6a | Lactose | 72 | 1 | 19.10 | 21.37 | 17.93 | 18.77 | 18.82 | 17.88 | 18.98 | 23.75 |
QM6a | Lactose | 72 | 2 | 19.67 | 20.73 | 17.88 | 19.15 | 19.25 | 18.20 | 19.37 | 23.40 |
QM6a | NaCl | 6 | 1 | 21.08 | 21.01 | 18.55 | 19.80 | 19.12 | 18.87 | 19.71 | n.m. |
QM6a | NaCl | 6 | 2 | 19.90 | 19.99 | 18.22 | 19.49 | 18.72 | 18.67 | 19.68 | n.m. |
QM6a | NaCl | 24 | 1 | 19.16 | 19.24 | 17.88 | 19.13 | 18.06 | 19.02 | 20.22 | n.m. |
QM6a | NaCl | 24 | 2 | 19.77 | 20.30 | 18.88 | 20.46 | 19.20 | 20.13 | 21.48 | n.m. |
QM6a | Xylan | 48 | 1 | 21.62 | 22.38 | 18.82 | 20.26 | 20.54 | 19.18 | 20.65 | n.m. |
QM6a | Xylan | 48 | 2 | 21.08 | 21.80 | 21.31 | 23.02 | 22.52 | 21.77 | 23.26 | n.m. |
QM6a | Xylan | 24 | 1 | 18.89 | 18.75 | 17.93 | 18.98 | 18.65 | 18.13 | 19.39 | n.m. |
QM6a | Xylan | 24 | 2 | 18.12 | 18.03 | 19.02 | 19.95 | 19.11 | 19.41 | 20.37 | n.m. |
Rut-C30 | Cellulose | 120 | 1 | 20.66 | 20.73 | 18.80 | 19.76 | 19.23 | 19.65 | 20.87 | 9.90 |
Rut-C30 | Cellulose | 120 | 2 | 20.32 | 21.33 | 17.61 | 18.34 | 17.84 | 18.24 | 19.26 | 9.90 |
Rut-C30 | Cellulose | 48 | 1 | 17.77 | 17.36 | 18.60 | 19.68 | 18.88 | 19.85 | 21.13 | 19.25 |
Rut-C30 | Cellulose | 48 | 2 | 17.69 | 17.36 | 18.38 | 19.40 | 18.61 | 19.46 | 20.56 | 16.90 |
Rut-C30 | DTT | 6 | 1 | 23.88 | 22.19 | 19.67 | 21.70 | 21.20 | 20.22 | 21.43 | 20.65 |
Rut-C30 | DTT | 6 | 2 | 25.49 | 25.82 | 19.11 | 21.24 | 20.32 | 18.49 | 19.45 | 20.85 |
Rut-C30 | DTT | 48 | 1 | 20.53 | 19.97 | 20.81 | 22.24 | 20.11 | 21.38 | 22.54 | 23.45 |
Rut-C30 | DTT | 48 | 2 | 20.96 | 20.73 | 16.79 | 17.86 | 17.63 | 17.31 | 18.56 | 23.45 |
Rut-C30 | Glucose | 84 | 1 | 23.04 | 23.98 | 18.54 | 19.39 | 19.08 | 19.24 | 20.59 | 21.75 |
Rut-C30 | Glucose | 84 | 2 | 23.84 | 24.92 | 18.76 | 19.82 | 19.93 | 19.20 | 20.50 | 19.10 |
Rut-C30 | Glucose | 24 | 1 | 18.62 | 18.82 | 18.53 | 19.33 | 18.91 | 19.37 | 20.43 | 26.50 |
Rut-C30 | Glucose | 24 | 2 | 18.58 | 18.39 | 19.56 | 20.64 | 20.07 | 20.54 | 22.19 | 26.55 |
Rut-C30 | Glycerin | 36 | 1 | 20.50 | 19.42 | 17.84 | 18.60 | 18.13 | 17.10 | 17.99 | n.m. |
Rut-C30 | Glycerin | 36 | 2 | 21.53 | 20.69 | 17.11 | 18.17 | 18.00 | 16.78 | 17.80 | n.m. |
Rut-C30 | Glycerin | 84 | 1 | 18.87 | 20.41 | 18.36 | 19.58 | 19.02 | 19.29 | 20.40 | n.m. |
Rut-C30 | Glycerin | 84 | 2 | 18.02 | 19.29 | 18.41 | 19.50 | 19.17 | 18.78 | 20.07 | n.m. |
Rut-C30 | Lactose | 48 | 1 | 19.96 | 20.20 | 21.54 | 22.46 | 21.24 | 21.55 | 23.05 | 16.40 |
Rut-C30 | Lactose | 48 | 2 | 19.68 | 20.86 | 20.21 | 21.25 | 21.03 | 20.33 | 21.88 | 14.40 |
Rut-C30 | Lactose | 72 | 1 | 21.74 | 22.29 | 18.18 | 19.11 | 19.37 | 18.23 | 19.39 | 25.20 |
Rut-C30 | Lactose | 72 | 2 | 20.01 | 22.33 | 18.75 | 20.07 | 19.98 | 18.87 | 20.17 | 18.70 |
Rut-C30 | NaCl | 6 | 1 | 20.38 | 19.90 | 17.73 | 19.20 | 18.14 | 18.77 | 19.73 | n.m. |
Rut-C30 | NaCl | 6 | 2 | 19.95 | 19.28 | 17.31 | 18.60 | 17.53 | 18.09 | 18.92 | n.m. |
Rut-C30 | NaCl | 24 | 1 | 20.13 | 19.27 | 18.10 | 19.34 | 18.44 | 18.56 | 19.35 | n.m. |
Rut-C30 | NaCl | 24 | 2 | 21.28 | 20.47 | 18.29 | 18.94 | 18.74 | 18.26 | 19.11 | n.m. |
Rut-C30 | Xylan | 48 | 1 | 21.95 | 21.70 | 17.57 | 18.67 | 18.59 | 17.65 | 18.80 | n.m. |
Rut-C30 | Xylan | 48 | 2 | 21.50 | 21.31 | 22.20 | 24.18 | 22.84 | 21.93 | 23.46 | n.m. |
Rut-C30 | Xylan | 24 | 1 | 20.05 | 18.94 | 20.10 | 21.32 | 20.61 | 19.67 | 21.04 | n.m. |
Rut-C30 | Xylan | 24 | 2 | 19.82 | 19.01 | 19.50 | 20.59 | 20.14 | 19.16 | 20.34 | n.m. |
Subgroup | Calculation Method | Gene | Rank | Calculated Value 1 |
---|---|---|---|---|
QM6a | DeltaCT | bzp1 | 1 | 1.310 |
tpc1 | 3 | 1.336 | ||
cue1 | 2 | 1.330 | ||
ubi1 | 4 | 1.374 | ||
sas3 | 5 | 1.396 | ||
act1 | 7 | 2.211 | ||
sar1 | 6 | 2.033 | ||
QM6a | BestKeeper | bzp1 | 1 | 0.92 |
tpc1 | 4 | 1.01 | ||
cue1 | 3 | 0.99 | ||
ubi1 | 2 | 0.95 | ||
sas3 | 5 | 1.10 | ||
act1 | 7 | 1.91 | ||
sar1 | 6 | 1.64 | ||
QM6a | Normfinder | bzp1 | 2 | 0.702 |
tpc1 | 3 | 0.840 | ||
cue1 | 1 | 0.547 | ||
ubi1 | 4 | 0.959 | ||
sas3 | 5 | 0.995 | ||
act1 | 7 | 2.017 | ||
sar1 | 6 | 1.753 | ||
QM6a | Genorm | ubi1/sas3 | 1/2 | 0.508 |
tpc1 | 3 | 0.717 | ||
bzp1 | 4 | 0.778 | ||
cue1 | 5 | 0.865 | ||
sar1 | 6 | 1.314 | ||
act1 | 7 | 1.570 | ||
Rut-C30 | DeltaCT | bzp1 | 1 | 1.213 |
cue1 | 2 | 1.226 | ||
tpc1 | 3 | 1.257 | ||
ubi1 | 4 | 1.359 | ||
sas3 | 5 | 1.457 | ||
act1 | 6 | 2.037 | ||
sar1 | 7 | 2.038 | ||
Rut-C30 | BestKeeper | ubi1 | 1 | 1.04 |
cue1 | 2 | 1.17 | ||
bzp1 | 3 | 1.20 | ||
sas3 | 4 | 1.20 | ||
tpc1 | 5 | 1.35 | ||
sar1 | 6 | 1.48 | ||
act1 | 7 | 1.56 | ||
Rut-C30 | Normfinder | cue1 | 1 | 0.527 |
bzp1 | 2 | 0.568 | ||
tpc1 | 3 | 0.725 | ||
ubi1 | 4 | 0.854 | ||
sas3 | 5 | 1.139 | ||
act1 | 6 | 1.823 | ||
sar1 | 7 | 1.826 | ||
Rut-C30 | Genorm | bzp1/tpc1 | 1/2 | 0.448 |
cue1 | 3 | 0.541 | ||
sas3 | 4 | 0.764 | ||
ubi1 | 5 | 0.823 | ||
act1 | 6 | 1.303 | ||
sar1 | 7 | 1.513 | ||
Early | DeltaCT | Tpc1 | 1 | 1.279 |
Bzp1 | 2 | 1.300 | ||
cue1 | 3 | 1.359 | ||
ubi1 | 4 | 1.462 | ||
sas3 | 5 | 1.582 | ||
act1 | 6 | 2.125 | ||
sar1 | 7 | 2.190 | ||
Early | BestKeeper | bzp1 | 1 | 0.93 |
ubi1 | 2 | 0.94 | ||
cue1 | 3 | 1.02 | ||
tpc1 | 4 | 1.06 | ||
sas3 | 5 | 1.19 | ||
sar1 | 6 | 1.43 | ||
act1 | 7 | 1.58 | ||
Early | Normfinder | tpc1 | 1 | 0.503 |
cue1 | 2 | 0.572 | ||
bzp1 | 3 | 0.637 | ||
ubi1 | 4 | 1.023 | ||
sas3 | 5 | 1.300 | ||
act1 | 6 | 1.882 | ||
sar1 | 7 | 1.987 | ||
Early | Genorm | bzp1/tpc1 | 1/2 | 0.609 |
ubi1 | 3 | 0.791 | ||
sas3 | 4 | 0.837 | ||
cue1 | 5 | 0.903 | ||
act1 | 6 | 1.383 | ||
sar1 | 7 | 1.614 | ||
Late | DeltaCT | bzp1 | 1 | 1.120 |
cue1 | 2 | 1.126 | ||
sas3 | 3 | 1.164 | ||
Ubi1 | 4 | 1.181 | ||
tpc1 | 5 | 1.236 | ||
sar1 | 6 | 1.698 | ||
act1 | 7 | 1.841 | ||
Late | BestKeeper | Ubi1 | 1 | 1.00 |
sar1 | 2 | 1.19 | ||
sas3 | 3 | 1.19 | ||
cue1 | 4 | 1.22 | ||
bzp1 | 5 | 1.23 | ||
tpc1 | 6 | 1.30 | ||
act1 | 7 | 1.52 | ||
Late | Normfinder | cue1 | 1 | 0.555 |
bzp1 | 2 | 0.607 | ||
sas3 | 3 | 0.722 | ||
Ubi1 | 4 | 0.737 | ||
tpc1 | 5 | 0.926 | ||
sar1 | 6 | 1.451 | ||
act1 | 7 | 1.663 | ||
Late | Genorm | bzp1/tpc1 | 1/2 | 0.574 |
cue1 | 3 | 0.638 | ||
sas3 | 4 | 0.766 | ||
ubi1 | 5 | 0.785 | ||
sar1 | 6 | 1.138 | ||
act1 | 7 | 1.339 |
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Growth Condition | Cultivation Volume (mL) | QM6a | Rut-C30 |
---|---|---|---|
Glucose | 200 | 36 h/84 h | 24 h/84 h |
Lactose | 200 | 72 h/96 h | 48 h/72 h |
Xylan | 200 | 24 h/48 h | 24 h/48 h |
Glycerin | 200 | 24 h/84 h | 36 h/84 h |
Cellulose | 200 | 48 h/120 h | 48 h/120 h |
Lactose-DTT | 50 | 6 h/48 h | 6 h/48 h |
Glucose-NaCl | 50 | 6 h/24 h | 6 h/24 h |
Gene Name | Primer Sequences (5′-3′) | Amplicon Length (bp) |
---|---|---|
act1 | Fwd: TGAGAGCGGTGGTATCCACG Rev: GGTACCACCAGACATGACAATGTTG | 103 |
sar1 | Fwd: TGGATCGTCAACTGGTTCTACGA Rev: GCATGTGTAGCAACGTGGTCTTT | 115 |
bzp1 | Fwd: GGCCTTTCTTTGAGCAGTGATG Rev: AGCTGCCCTTTGTTGTTGTC | 92 |
tpc1 | Fwd: TATGCGAATGAGCCGATTCC Rev: AACGTCCAGCTTCACATTGG | 78 |
cue1 | Fwd: GCGTAATCAAGGCGGTTCTG Rev: TGTTTTGCGGCTCGTTCTTG | 108 |
ubi1 | Fwd: TCAAATGCGGGCGACAAAAG Rev: TGTTGACCGGATGTTTGCAC | 112 |
sas3 | Fwd: ATCGCGTGCTGTACATTTGC Rev: TGTTTCGCAGCGCATTTGAG | 91 |
cbh1 | Fwd: ACTATGTCCAGAATGGCGTC Rev: TGGCGTAGTAATCATCCC | 209 |
Gene Name | Gene Description | Transcript ID | CV (QM6a) | CV (Rut-C30) |
---|---|---|---|---|
bzp1 | B-ZIP domain protein | TRIREDRAFT_50536 | 0.1450 | 0.1038 |
tpc1 | Trafficking protein particle complex subunit 1 | TRIREDRAFT_49838 | 0.2169 | 0.0993 |
cue1 | CUE domain-containing protein | TRIREDRAFT_29932 | 0.2743 | 0.1691 |
ubi1 | ubiquitin-like 1-activating enzyme E1 B | TRIREDRAFT_61945 | 0.1367 | 0.1340 |
sas3 | Histone acetyltransferase SAS3 | TRIREDRAFT_5916 | 0.1787 | 0.1237 |
act1 | Actin | TRIREDRAFT_44504 | 0.3725 | 0.3534 |
sar1 | Secretion-associated Ras-related GTPase | TRIREDRAFT_61470 | 0.9250 | 0.6846 |
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Danner, C.; Karpenko, Y.; Mach, R.L.; Mach-Aigner, A.R. Act1 out of Action: Identifying Reliable Reference Genes in Trichoderma reesei for Gene Expression Analysis. J. Fungi 2025, 11, 396. https://doi.org/10.3390/jof11050396
Danner C, Karpenko Y, Mach RL, Mach-Aigner AR. Act1 out of Action: Identifying Reliable Reference Genes in Trichoderma reesei for Gene Expression Analysis. Journal of Fungi. 2025; 11(5):396. https://doi.org/10.3390/jof11050396
Chicago/Turabian StyleDanner, Caroline, Yuriy Karpenko, Robert L. Mach, and Astrid R. Mach-Aigner. 2025. "Act1 out of Action: Identifying Reliable Reference Genes in Trichoderma reesei for Gene Expression Analysis" Journal of Fungi 11, no. 5: 396. https://doi.org/10.3390/jof11050396
APA StyleDanner, C., Karpenko, Y., Mach, R. L., & Mach-Aigner, A. R. (2025). Act1 out of Action: Identifying Reliable Reference Genes in Trichoderma reesei for Gene Expression Analysis. Journal of Fungi, 11(5), 396. https://doi.org/10.3390/jof11050396