Total RNA was extracted from G. lucidum hyphae using RNAiso Plus (TaKaRa, Dalian, China) as described in a previous study (41 (link)). A 5× All-In-One RT MasterMix kit (TaKaRa) was used to obtain the cDNA. G. lucidum hyphae were ground into powder with liquid nitrogen, and DNA was extracted by the CTAB method. Quantitative real-time RT-PCR (qRT-PCR) analysis was performed using the EvaGreen 2× qPCR MasterMix kit (ABM, Zhenjiang, China) with the primers listed in Table S1. The gene fragments were amplified by real-time PCR using primers based on the G. lucidum genome sequence with the primers shown in Table S1. The GlAZ mutant strain expression was evaluated by calculating the difference between the threshold cycle (CT) value of the gene analyzed and the CT value of the housekeeping gene 18S rRNA with the primers listed in Table S1. Quantitative reverse transcription-PCR (qRT-PCR) calculations analyzing the relative gene expression level were performed according to the 2-ΔΔCT method as described in a previous study (42 (link)).
All in one rt mastermix kit
Manufactured by Takara Bio
The 5× All-In-One RT MasterMix kit is a laboratory reagent designed for reverse transcription and subsequent amplification of RNA samples. It contains all the necessary components for these processes in a single, concentrated solution.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso plus, by Takara Bio (2 mentions)
Evagreen 2 qpcr mastermix kit, by Applied Biological Materials (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanovue spectrophotometer, by GE Healthcare (1 mentions)
3 protocols using all in one rt mastermix kit
1
Quantitative Analysis of G. lucidum Hyphal Transcript Levels
2
Quantitative Analysis of mRNA Expression
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Total RNA from cells and colon tissues was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA). NanoVue spectrophotometer (GE Healthcare) was used to assess the quantity and quality. mRNA was subjected to reverse transcription with 5′All-In-One RT MasterMix kit (TaKaRa). For quantitative analysis of mRNA expression, real-time PCR was performed using StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). β-actin was used as an internal control for mRNA expression. 2-△△Ct method was used to quantify the relative expression levels. The primers used for the real-time PCR analysis were summarized in Table S6 .
3
Quantitative Analysis of G. lucidum Hyphal Transcript Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from G. lucidum hyphae using RNAiso Plus (TaKaRa, Dalian, China) as described in a previous study (41 (link)). A 5× All-In-One RT MasterMix kit (TaKaRa) was used to obtain the cDNA. G. lucidum hyphae were ground into powder with liquid nitrogen, and DNA was extracted by the CTAB method. Quantitative real-time RT-PCR (qRT-PCR) analysis was performed using the EvaGreen 2× qPCR MasterMix kit (ABM, Zhenjiang, China) with the primers listed in Table S1. The gene fragments were amplified by real-time PCR using primers based on the G. lucidum genome sequence with the primers shown in Table S1. The GlAZ mutant strain expression was evaluated by calculating the difference between the threshold cycle (CT) value of the gene analyzed and the CT value of the housekeeping gene 18S rRNA with the primers listed in Table S1. Quantitative reverse transcription-PCR (qRT-PCR) calculations analyzing the relative gene expression level were performed according to the 2-ΔΔCT method as described in a previous study (42 (link)).
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