Evaluation of Reference Genes for RT-qPCR Studies in the Seagrass Zostera muelleri Exposed to Light Limitation

Abstract

Seagrass meadows are threatened by coastal development and global change. In the face of these pressures, molecular techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) have great potential to improve management of these ecosystems by allowing early detection of chronic stress. In RT-qPCR, the expression levels of target genes are estimated on the basis of reference genes, in order to control for RNA variations. Although determination of suitable reference genes is critical for RT-qPCR studies, reports on the evaluation of reference genes are still absent for the major Australian species Zostera muelleri subsp. capricorni (Z. muelleri). Here, we used three different software (geNorm, NormFinder and Bestkeeper) to evaluate ten widely used reference genes according to their expression stability in Z. muelleri exposed to light limitation. We then combined results from different software and used a consensus rank of four best reference genes to validate regulation in Photosystem I reaction center subunit IV B and Heat Stress Transcription factor A- gene expression in Z. muelleri under light limitation. This study provides the first comprehensive list of reference genes in Z. muelleri and demonstrates RT-qPCR as an effective tool to identify early responses to light limitation in seagrass.

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues

“…The literature is constellated by studies focusing on the selection of reference genes in different plant species, tissues, conditions [ 1 , 2 , 3 , 4 ]; a list of potential candidates whose orthologs can be tested in the plant/tissue of choice is usually provided. Several types of software are available to assess the stability of a set of candidate reference genes [ 5 , 6 , 7 , 8 ] and a ranking is provided to show those most stable.…”

Low oxygen affects photophysiology and the level of expression of two-carbon metabolism genes in the seagrass Zostera muelleri

“…The corresponding CT values were used directly in the software package NormFinder (Andersen et al 2004) to rank and select the most stable reference genes. Candidate reference genes and corresponding primers used in this study were identified previously (Schliep et al 2015). Because these candidates reference genes were initially validated under low light stress conditions, we used NormFinder to measure their stability value during low O2 exposure (i.e.…”

Molecular physiology reveals ammonium uptake and related gene expression in the seagrass Zostera muelleri

“…To validate changes in target genes expression, CT were imported into the qbase+ software package (Biogazelle). Expression data for target genes was first normalized against the best combination of 3 reference genes as previously described (Adenosylhomocysteinase, AHCY; Glyceraldehyde 3-phosphate dehydrogenase, GADPH; and Translation initiation factor 1 subunit beta, Elof1; Supplementary Table S1, Supplementary Figure S1) (Schliep et al, 2015) using GeNorm algorithm within qbase+ software package (Biogazelle) and then transformed into quantities using maximum efficiency of 1.00 (or 100%) to obtain Calibrated Normalized Relative Quantities (CNRQ) (Hellemans et al, 2007).…”

“…With the future advances of digital qPCR, the use of these markers might not even require the step of housekeeping genes selection that is normally needed to normalize RT-qPCR data (Schliep et al, 2015, Zmienko et al, 2015. Consequently, as long as a threshold database can be established and maintained for different species, our results may have implications for the management of seagrass meadows.…”