Evaluation of reference genes for human chondrocytes cultured in several different thermal environments.
OBJECTIVE
The purpose of this study was to identify reference genes showing stable expression in chondrocytes cultured under several different thermal environments and in different culture systems.


METHODS
Human articular chondrocytes were cultured by monolayer or pellet culture system at 32 °C, 37 °C, and 41 °C for 3 days. Thereafter, the total RNA was extracted, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed. The qRT-PCR data was analysed using three different algorithms (geNorm, NormFinder, and BestKeeper) to identify reference genes exhibiting stable expression from among the seven candidate reference genes (B2M, ACTB, GAPDH, HSPCB, RPL13a, YWHAZ, and 18S).


RESULTS
The candidate reference genes, except for HSPCB and YWHAZ, showed systematic variations in expression. In the monolayer culture, RPL13a was the most stable gene identified using NormFinder and BestKeeper; on using geNorm, ACTB and GAPDH showed the highest expression stability. In the pellet culture, ACTB was the most stable gene identified using NormFinder and BestKeeper, whereas GAPDH and RPL13a were the most stable reference genes as determined using geNorm. In the combined group, B2M and GAPDH were the most stable genes identified using geNorm, whereas RPL13a and YWHAZ were the most stable as per NormFinder and BestKeeper, respectively. The best combination of two candidate reference genes among all the groups determined using NormFinder was RPL13a and YWHAZ.


CONCLUSIONS
The combination of RPL13a and YWHAZ might be suitable as reference genes for human chondrocytes cultured at 32 °C, 37 °C, and 41 °C in monolayer, pellet, or combined cultures.
Department of Motor Function Analysis, Human Health Sciences, Graduate School of Medicine, Kyoto University , Kyoto .
Akira
A
Ito
Tomoki
T
Aoyama
Junichi
J
Tajino
Momoko
M
Nagai
Shoki
S
Yamaguchi
Hirotaka
H
Iijima
Xiangkai
X
Zhang
Haruhiko
H
Akiyama
Hiroshi
H
Kuroki
D001483:Base Sequence; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D017931:DNA Primers