RESUMO
OBJECTIVE:To establish a method of simultaneous determination of inulicin and deacetylinulicin in Inulae Flos. METHODS:HPLC was performed on the column of Zorbax SB-C18 with mobile phase of acetonitrile-water(gradient elution)at a flow rate of 1.0 ml/min,the column temperature was 25 ℃,the detection wavelength was 210 nm,and the injection volume was 10 μl. RESULTS:The linear range was 0.000 2-0.005 μg/ml(r=0.999 8)for inulicin and 0.000 1-0.001 7 μg/ml(r=0.999 4)for deacetylinulicin;RSDs of precision,stability and reproducibility tests were lower than 2.0%;recoveries were 99.63%-103.56%(RSD=1.26%,n=9)and 95.98%-101.21%(RSD=1.84%,n=9),respectively. CONCLUSIONS:The method is simple,accurate and reliable,and can be used for the quality evaluation of Inulae Flos.
RESUMO
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique widely used for quantification of mRNA transcription. Data normalization is an indispensable process for RT-qPCR and reference genes are most commonly used to normalize RT-qPCR and to reduce possible errors generated in the quantification of genes among several proposed methods. To date, RT-qPCR has been used in terms of gene expression studies in black rockfish (Sebastes schlegeli) but the majority of published RT-qPCR studies still lack proper validation of the reference genes. In the present study, mRNA transcription profiles of eight putative reference genes (18S rRNA, ACTB, GAPDH, TUBA, RPL17, EF1A, HPRT, and B2M) were examined using RT-qPCR in different tissues and larvae developmental stages of black rockfish. Three common statistical algorithms (geNorm, NormFinder, and BestKeeper) were used to assess expression stability and select the most stable genes for gene normalization. Two reference genes, RPL17 and EF1A showed high stability in black rockfish tissue analysis, while GAPDH was the least stable gene. During larvae developmental stages, EF1A, RPL17 and ACTB were identified as the optimal reference genes for data normalization, whereas B2M appeared unsuitable as the reference gene. In summary, our results could provide a useful guideline for reference gene selection and enable more accurate normalization of gene expression data in gene expression studies of black rockfish.