Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress

Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes,TUB, TUA, GADPH, EF1, 18S, GTP, ACT, UBI, UBC,andH2A, were tested for their adequacy inLentinula edodes(L. edodes). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia ofL. edodes, which were treated with high-temperature stress at 37°C for 0, 4, 8, 12, 18, and 24 hours. After treatment, expression stability of candidate genes was evaluated using three statistical software programs: geNorm, NormFinder, and BestKeeper. According to geNorm,TUBhad the lowest M values inL. edodesstrains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 wasTUB(0.030), and the best candidate reference gene in strain 18N44 wasUBI(0.047). In BestKeeper analysis, the standard deviation (SD) values ofUBC,TUA,H2A,EF1,ACT,18S, andGTPin strain 18 and those ofGADPHandGTPin strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, onlyUBIandTUBwere found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses,TUBwas the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction inL. edodesmycelia under high-temperature stress.

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Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

10.21203/rs.3.rs-35251/v3 ◽

2020 ◽

Author(s):

Huiyun Song ◽

Wenmai Mao ◽

Zhihao Duan ◽

Qingmin Que ◽

Wei Zhou ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Accurate Method ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Toona Ciliata

Abstract Background:Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem ( T. ciliata ). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions.Results:The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta ( H. robusta ) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 ( TcMYB3) gene.Conclusions:This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.

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