Selection of reference genes for expression analysis of plant-derived microRNAs in Plutella xylostella using qRT-PCR and ddPCR

PloS One
Lingling ZhangMinsheng You

Abstract

The establishment of an expression quantification system that can be easily applied for the comparison of microRNAs (miRNAs) from biological samples is an important step toward understanding functional mechanisms in organisms. However, there is lack of attention on the selection of reference genes for miRNA expression profiling in insect herbivores. Here, we explored the candidate reference genes in a notorious pest of cruciferous crops, Plutella xylostella, for normalization of miRNA expression in developmental stages and tissues and in response to a change of food source from artificial diet to host plant Arabidopsis thaliana. We first compared the expression levels and stability of eight small RNAs using qRT-PCR, and found that miR11 was the most suitable reference gene for expression quantification of the miRNAs. We then confirmed this finding using digital droplet PCR and further validated with a well-studied cross-kingdom miRNA derived from A. thaliana (ath-miR159a). However, none of the reference genes was applicable for all experimental conditions, and multiple reference genes were sometimes required within the same experiment. Our work provides a method for the selection of reference genes for quantification of plant-d...Continue Reading

References

May 16, 2001·Molecular and Cellular Probes·H J HnatyszynG Kraus
Jun 26, 2001·Journal of Biochemical and Molecular Toxicology·N J Walker
Aug 6, 2002·Proceedings of the National Academy of Sciences of the United States of America·Andreas RatzkaJuergen Kroymann
Apr 9, 2005·Genes and Immunity·J HuggettA Zumla
Nov 30, 2005·Nucleic Acids Research·Caifu ChenKarl J Guegler
Jun 23, 2006·Nature Methods·Frank DiehlDevin Dressman
Apr 5, 2007·Nature Protocols·Tania NolanStephen A Bustin
Feb 15, 2011·Phytochemistry·Inis Winde, Ute Wittstock
Nov 1, 2011·Analytical Chemistry·Benjamin J HindsonBill W Colston
Oct 2, 2012·Annual Review of Entomology·Michael J FurlongLloyd M Dosdall
Oct 30, 2012·Insect Biochemistry and Molecular Biology·Sassan Asgari
Sep 5, 2013·Clinical Chemistry·Tanis C DingleKeith R Jerome
Apr 18, 2014·Methods in Molecular Biology·Jan Hellemans, Jo Vandesompele
Dec 3, 2014·Biochemical and Biophysical Research Communications·Mengqin XiangXi Zeng
Dec 5, 2014·Cancer Epidemiology, Biomarkers & Prevention : a Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology·Elena MiottoManuela Ferracin
Dec 17, 2015·Annual Review of Entomology·Zhenyu LiMichael J Furlong
Aug 27, 2016·Biomedit︠s︡inskai︠a︡ khimii︠a︡·Y Y KiselevaA I Archakov
Dec 28, 2016·International Journal of Molecular Sciences·Anna Lukasik, Piotr Zielenkiewicz

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

RefFinder
SPSS Statistic
geNorm
Delta CT
NormFinder
Delta
BestKeeper

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.