scholarly journals Identification of Suitable Reference Genes for RT-qPCR Assays in Liriodendron chinense (Hemsl.) Sarg

Forests ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 441 ◽  
Author(s):  
Zhonghua Tu ◽  
Ziyuan Hao ◽  
Weiping Zhong ◽  
Huogen Li
Keyword(s):  
Reference Genes ◽  
Target Gene ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Stability ◽  
Chain Reaction ◽  
Multiple Organs ◽  
Liriodendron Chinense ◽  
Polymerase Chain ◽  
The Difference ◽  
Suitable Reference

The precision and reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) depend mainly on suitable reference genes; however, reference genes have not yet been identified for Liriodendron chinense (Hemsl.) Sarg. In this study, the expression stability of 15 candidate reference genes, ACT7, ACT97, UBQ1, eIF2, eIF3, HIS, BIG, AGD11, EFG, GAPDH, CYP, RPL25, UBC, RPB1, and TUB, was tested across multiple organs of L. chinense using four algorithms, geNorm, NormFinder, BestKeeper, and RefFinder. To understand the difference between the selected reference genes and the unsuitable candidate reference genes, the expression level of a target gene, LcPAT7, was normalized across various plant samples. ACT97 and eIF3 represented the best combination across all samples tested, while AGD11 and UBQ1 were unsuitable for normalization in this case. In the vegetative organ subset, ACT97, ACT7, and GAPDH showed the highest expression stability. For floral organs, UBC and eIF3 were the most stable reference genes. Unsuitable reference genes underestimated the expression levels of a target gene, LcPAT7. This study identified two reference genes (ACT97 and eIF3) for the precise and reliable normalization of L. chinense RT-qPCR data across various organs. Our work provides an effective framework for quantifying gene expression in L. chinense.

scholarly journals Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress

2021 ◽  
Vol 12 ◽  
Author(s):  
Ning Zhao ◽  
Junnan Xu ◽  
Lingxia Jiao ◽  
Mengzhen Qiu ◽  
Jie Zhang ◽  
...  
Keyword(s):  
16S Rrna ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Target Gene ◽  
Fruit Juice ◽  
Quantitative Polymerase Chain Reaction ◽  
Acid Stress ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
First Time

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI, dnaG, and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytvI, dnaG, and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.

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