Reference gene validation for normalization of RT-qPCR assay associated with germination and survival of rice under hypoxic condition

Kumar, Dhananjay; Das, Prasanta Kumar; Sarmah, Bidyut Kumar

doi:10.1007/s13353-018-0466-1

Research article2018Peer reviewedOpen access

Reference gene validation for normalization of RT-qPCR assay associated with germination and survival of rice under hypoxic condition

Kumar, Dhananjay; Das, Prasanta Kumar; Sarmah, Bidyut Kumar

Abstract

Study on expression of genes for the traits associated with hypoxia tolerance during the germination demands robust choice of reference genes for transcript data normalization and gene validation through real-time quantitative polymerase chain reaction (RT-qPCR). However, reliability and stability of reference genes across different rice germplasms under hypoxic condition have not been accessed yet. Stability performance of reference genes such as eukaryotic elongation factor 1 alpha (eEF1 alpha), ubiquitin 10 (UBQ10), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18SrRNA), 25S ribosomal RNA (25SrRNA), beta-tublin (beta-TUB), actin11 (ACT11), ubiquitin C (UBC), eukaryotic elongation factor 4 alpha (eIF4 alpha), and ubiquitin5 (UBQ5) was accessed through statistical algorithms like geNorm, NormFinder, Comparative Delta Ct method(,) BestKeeper, and RefFinder in three rice germplasms (KHO, RKB, and IR-64) with varied level of tolerance to hypoxic condition during germination. Among all genes used, OsGAPDH was found to be the most suitable reference gene under hypoxic condition. The performance of the highest-ranking reference gene (OsGAPDH) in terms of stability based on statistical algorithms was further validated for its reliability and stability through RT-qPCR with hypoxia-induced target gene OsTTP7. The identified stable housekeeping gene could be used as internal control for gene expression analysis in rice under hypoxia.

Keywords

Oryzasativa L; RT-qPCR; Hypoxia; RefFinder; OsGAPDH

Published in

Journal of Applied Genetics
2018, volume: 59, number: 4, pages: 419-430
Publisher: SPRINGER HEIDELBERG

SLU Authors

Kumar, Dhananjay
- Department of Plant Breeding, Swedish University of Agricultural Sciences
- Assam Agricultural University (AAU)

UKÄ Subject classification

Plant Biotechnology

Publication identifier

DOI: https://doi.org/10.1007/s13353-018-0466-1

Permanent link to this page (URI)

https://res.slu.se/id/publ/96912