Development and validation of endogenous reference genes for expression profiling of medaka (Oryzias latipes) exposed to endocrine disrupting chemicals by quantitative real-time RT-PCR.
The quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR) technique has been increasingly used in endocrine disrupting chemicals (EDCs) research. Usually, an appropriate endogenous control gene is critical for Q-RT-PCR to normalize the errors and sample-to-sample variations that occur in the course of tissue collection, RNA isolation, and RT-PCR. In this study, we cloned ribosomal protein L7 (RPL-7) from medaka (Oryzias latipes), and then used Q-RT-PCR to study its transcription characteristics and those of glyceraldehyde-3-phosphate dehydrogenase, beta-actin, mitochondrial 16S ribosomal RNA (16S rRNA), and 18S rRNA. Of the five genes, RPL-7 and 18S rRNA were expressed with the less variance among the same tissue samples, in different tissues, and stages of development and were unaffected by EDCs exposure. The expression levels of RPL-7 among different tissues were between 9.76 x 10(6) +/- 9.49 x 10(5) and 1.39 x 10(7) +/- 1.69 x 10(6) copies/microg RNA but those of 18S rRNA were as high as 4.48 x 10(11) +/- 5.95 x 10(10) to 5.90 x 10(11) +/- 1.21 x 10(10) copies/microg RNA, which is above the usual detection scope of Q-RT-PCR if no complementary DNA reaction dilution is performed. As a result, RPL-7 is the single suitable endogenous control gene for expression profiling in future studies, especially in studies on the EDCs issue using medaka.
Zhang
Zhaobin
Z
College of Environmental Science, Peking University, Beijing 100871, China.
Hu
Jianying
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D052244:Endocrine Disruptors / Q000633:toxicity*; D015870:Gene Expression / Q000187:drug effects*; D020869:Gene Expression Profiling; D008969:Molecular Sequence Data; D009928:Organ Specificity; D009990:Oryzias / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics; D014158:Transcription, Genetic
D018076:DNA, Complementary; D052244:Endocrine Disruptors; D012269:Ribosomal Proteins