Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii.

Hucheng Jiang,Hui Wang,Wei Lu,Huaiyu Ding,Hongwei Yu,Zhaojun Qian,Jiale Li

doi:10.3390/ijms160921591

Abstract

qRT-PCR is a widely used technique for rapid and accurate quantification of gene expression data. The use of reference genes for normalization of the expression levels is crucial for accuracy. Several studies have shown that there is no perfect reference gene that is appropriate for use in all experimental conditions, and research on suitable reference genes in red swamp crawfish (Procambarus clarkii) is particularly scarce. In this study, eight commonly used crustacean reference genes were chosen from P. clarkii transcriptome data and investigated as potential candidates for normalization of qRT-PCR data. Expression of these genes under different experimental conditions was examined by qRT-PCR, and the stability of their expression was evaluated using three commonly used statistical algorithms, geNorm, NormFinder and BestKeeper. A final comprehensive ranking determined that EIF and 18S were the optimal reference genes for expression data from different tissues, while TBP and EIF were optimal for expression data from different ovarian developmental stages. To our knowledge, this is the first systematic analysis of reference genes for normalization of qRT-PCR data in P. clarkii. These results will facilitate more accurate and reliable expression studies of this and other crustacean species.

Highlights

The red swamp crawfish Procambarus clarkii is a freshwater species native to the South-CentralUnited States and Northeastern Mexico but has spread in range to become one of the most invasive species worldwide [1,2,3]
The results showed that the primer efficiency of the eight reference genes ranged from 96.0% to 103.6%, and the correlation coefficient (R2) values ranged from 0.992 to 0.999 (Table 1)
Cycle Threshold (Ct) values ranged from 15.11 to 30.48 overall, from 15.36 (GAPDH) to 30.48 (TBP) for samples in different tissues (Figure 1A) and from 15.11 (ACTB) to 30.04 (EF-1α) for samples in different ovarian developmental stages (Figure 1B). These results showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ACTB were the most abundant transcripts with the lowest Ct values, whereas TATA-binding protein (TBP) and elongation factor 1α (EF-1α) had the lowest expression levels with the highest Ct values

Summary

Introduction

The red swamp crawfish Procambarus clarkii is a freshwater species native to the South-CentralUnited States and Northeastern Mexico but has spread in range to become one of the most invasive species worldwide [1,2,3]. The red swamp crawfish Procambarus clarkii is a freshwater species native to the South-Central. P. clarkii was introduced from Japan to Nanjing, China in 1929 [4,5], and this species has been farmed extensively in China since the 1990s, which is the world’s leading crawfish producer [6,7]. In order to develop novel methods for controlling gonad maturation in crawfish to improve yields, it is critical to understand the molecular mechanisms of ovarian development and oogenesis [9]. Oogenesis is a complicated differentiation process regulated by a vast number of intra- and extra-ovarian factors [10,11,12]. Understanding gene expression is essential for elucidating the functional mechanisms underlying this complex process. Many factors can affect the accuracy of qRT-PCR, including the quality of the RNA, the efficiency of reverse transcription, the primer specificity and amplification efficiency, and the statistical analysis methods employed [15]

Methods

Results

Conclusion