Selection and evaluation of housekeeping genes for haemocytes of soft-shell clams (Mya arenaria) challenged with Vibrio splendidus.
Gene expression studies have opened a tremendous field of investigation in biological research over the last decades. Expression of genes is most frequently quantified by real time PCR (RT-qPCR), as this method has proven to be highly sensitive. One of the critical steps, however, in comparing transcription profiles is the availability of selected housekeeping genes. Expression of these genes should be steadily stable across the conditions under study so that they provide a baseline for gene expression comparison. Such a baseline is best established using a set of few housekeeping genes. Usually, those genes are involved in maintaining homeostasis and cell viability. In our study, nine candidate genes were used, including some commonly used housekeeping genes, such as ribosomal RNA (18S, S-15, S-18 and L-37), beta actin, ubiquitin, receptor activated C kinase (RACK) and elongation factor 1 and 2, in order to determine the most stable housekeeping genes, after haemocytes of Mya arenaria were exposed to Vibrio splendidus for 2 h. Our results showed that EF-1, S-18 and ubiquitin appear to be the most stable genes for this experimental condition. On the other hand, both 18S and beta actin, the most widely used housekeeping genes, turned out to be the least stable. This demonstrates the absolute need for preliminary assessment of housekeeping genes in gene expression studies.
Araya
Mebrahtu T
MT
University of Prince Edward Island, Atlantic Veterinary College, Department of Pathology and Microbiology, Charlottetown, Prince Edward Island, Canada C1A 4P3. mebrahtut2001@yahoo.com
Siah
Ahmed
A
Mateo
Dante
D
Markham
Frederick
F
McKenna
Patty
P
Johnson
Gerry
G
Berthe
Franck C J
FC
D000818:Animals; D001483:Base Sequence; D002470:Cell Survival; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D042822:Genomic Instability; D006434:Hemocytes / Q000166:cytology / Q000382:microbiology / Q000502:physiology*; D006706:Homeostasis; D008969:Molecular Sequence Data; D049892:Mya / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D012335:RNA, Ribosomal / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014733:Vibrio / Q000502:physiology*; D014735:Vibrio Infections / Q000235:genetics
D012335:RNA, Ribosomal