Total RNA of T. rubrum was extracted from five samples of the 256 μg/ml group and five samples of the control group using the Rnaiso plus kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. The concentration and purity of total RNA were assessed by ultramicro-spectrophotometers (NanoDrop 2000, Thermo Fisher Scientific, Wilmington, DE, United States) at the absorbance ratios of A260/230 and A260/280. Subsequently, the first strand of cDNA was synthesized from 2 μg of total RNA using the FastQuant RT Kit (with gDNase) (Takara Bio, Shiga, Japan) and was stored at −20°C.
Fastquant rt kit with gdnase
Manufactured by Takara Bio
Sourced in Japan, China
The FastQuant RT kit with gDNase is a laboratory equipment product designed for the reverse transcription of RNA. It includes a gDNase component for the removal of genomic DNA before the reverse transcription step.
Automatically generated - may contain errors
5 protocols using fastquant rt kit with gdnase
1
Total RNA Extraction and cDNA Synthesis from T. rubrum
2
Quantification of Fungal Gene Expression
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The WT strain and OE-275 mutant were cultured in PDA medium at 28 °C for 6 days. The vegetative hyphae were harvested in 9-cm Petri dishes, which were then incubated at 28 °C for 3 and 5 d. After induction, hyphae were collected and frozen immediately in liquid nitrogen. Total RNA was extracted from all samples with the AxyPrep Multisource RNA Miniprep Kit (Axygen, Jiangsu, China). The extracted RNA was then reverse transcribed to cDNA with the FastQuant RT Kit with gDNase (Takara, Kusatsu, Japan). The cDNA was used as the template for a qRT-PCR assay, which was conducted to analyze the transcription levels of candidate genes. The gene-specific qRT-PCR primers were designed with Primer3 software, and the β-tubulin gene (Tub, AOL_s00076g640) was used as an internal standard. The qRT-PCR analysis was performed as previously described [16 (link)]. The relative transcription level (RTL) of each gene was calculated as the ratio of the transcription level in the deletion mutant to the transcription level in the WT strain at a given timepoint according to the 2−ΔΔCt method [16 (link)].
3
Quantifying Conidiation and Stress Response
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WT and Δfus3 strains were cultured on TYGA at 28°C for 3, 5, or 7 days, and total RNA from mycelium was extracted with AxyPrep multisource RNA miniprep kit, and then translated into cDNA a FastQuant RT kit with gDNase (TaKaRa).The cDNA was further used as a template for RT-qPCR using SYBR Premix Ex TaqTM (TaKaRa) and specific primers (Table S1 ) to detect the transcript levels of candidate genes related to conidiation, cell wall biosynthesis, and oxidative stress response; the β-tubulin-encoding gene was used as an internal standard. The mRNA level was analyzed using the 2−ΔΔCt method.51 (link)
4
Quantification of Sporulation Genes in Mutant Strains
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To analyze the expression of sporulation-related genes in WT and mutant strains, total RNAs were extracted from 3-, 5-, and 7-day cultures grown on TYGA by using an AxyPrep multisource RNA miniprep kit (Axygen, Jiangsu, China), and then reverse-transcribed into cDNAs by using a FastQuant RT kit with gDNase (TaKaRa). The cDNA samples of each strain were used as the template to assess the transcript level of each gene by using SYBRH Premix Ex Taq (TaKaRa) and performing RT-PCR with paired primers (Supplementary Table S2 ); β-tubulin served as an internal standard, and the 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used for quantification. The relative transcription level (RTL) of each gene was calculated as the ratio of the transcription level in the deletion mutant to the transcription level in the WT strain at a given time point.
5
Transcriptional Profiling of A. oligospora
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The hyphae of the WT and mutant strains were cultured on TYGA at 28°C, and the mycelia samples were collected on the 3rd, 5th, and 7th day, and the total RNA of mycelia samples was isolated using an AxyPrep multisource RNA miniprep kit (Axygen, Jiangsu, China), and then reverse-transcribed into cDNA using a FastQuant RT kit with gDNase (TaKaRa). The cDNA samples of each strain were used as templates for RT-PCR analysis with specific paired primers (Supplementary Table S2 ). The β-tubulin gene of A. oligospora was used as an internal standard, and the transcriptional level of the genes associated with conidiation and cell wall synthesis was calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link)). Three replicates were performed in the RT-PCR experiments, and the RT-PCR analysis for each gene was repeated three times. The relative transcript level (RTL) of candidate genes was estimated as the transcript ratio of each mutant vs. the WT.
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