Identification of the appropriate reference gene through using a realtime quantitative PCR in grapes

【Objective】A real-time quantitative PCR(q RT-PCR) is an efficient and reliable molecular technique to determine changes in a gene's m RNA expression. Compared with earlier methods, q RTPCR has the advantages of high speed, high sensitivity, high degrees of automation and reproducibility.The most common procedure used for q RT-PCR is the relative measurement of gene expression of interest after normalization using endogenous reference genes. The selection of suitable reference genes for q RTPCR is important for accurate normalization of the target gene expression found in specific experimental materials or conditions. Although the reference gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene. Beginning in 2002, ge Norm has been developed to select stably expressed reference genes, and to use multiple reference genes for normalization in q RT-PCR. ge Norm determines the expression stability of candidate reference genes by gene stability measure value(M). ge Norm determines the number of candidate reference genes through using a pairwise variation V value. Reference genes are selected and analyzed by using ge Norm to calculate the target gene expression in various tissues of different grape cultivars.【Methods】Experiment Material consisted of eight different grape cultivars including:‘Jufeng'‘Jumeigui'‘Zuijinxiang'‘Shenfeng'‘Shenyu'‘Shenhua'‘Xiahei'and‘Hupei 1#'. Total RNA was extracted using a RNA extraction kit(Omega) according to the manufacturer's instructions, and the DNA was digested at 37 °C for 30 min using DNase I(Takara) according to the manufacturer's instructions. The concentration of each RNA sample was determined using a spectrophotometer. Only samples with an absorbance ratio at 260 and 280 nm(A260/A280) ≥ 1.9 were used. To obtain the first-strand c DNA, 1 μg of each DNA-free RNA sample was used for reverse transcription with the M-MLV reverse transcriptase kit(Takara). The quantitative measurement of the gene expression was performed using the c DNA samples with a Lighe Cycler 480 System(Roche) and the SYBR Green fluorescence dye(Takara). In our present study, six commonly used housekeeping genes, Vv GAPDH, Vv UBQ, Vv ACT1, Vv EF1 r, Vv EF1-α andVv ACTIN, were chosen. The sequences of primers for the six candidate reference genes were cites with their appropriate references. The expression of the six candidate reference genes is assessed by using the q RT-PCR in the leaves and peels of the eight grape cultivars. The cycling conditions included 1 cycle at95 ℃ for 30 s, followed by 40 cycles of amplification at 95 ℃ for 5 s, and 60 ℃ for 34 s. All the candidate reference genes were able to specifically amplify with high efficiency in the q RT-PCR reaction. The slope of the standard curve is maintained at approximately-3.34. To determine the expression stability of the reference genes, q RT-PCR data were analyzed using a common available software, ge Norm. The internal control gene-stability measure M has been defined as the average pairwise variation of a particular gene with all other reference genes. Genes with the lowest M values have the most stable expression. A large variation means that the added gene has a significant effect and should preferably be included for calculation of a reliable normalization factor. Based on the Genome Biology data, 0.15 is proposed as a cut-off value, below which the inclusion of an additional reference gene is not required. For example, if the V3/4value is 0.21, then the normalization factor should preferably contain at least the 4 best reference genes.Subsequently, if the V4/5value is 0.13, then there is no real need to include a 5thgene as part of the normalization factor. An optimal number of reference genes for normalization in this example would therefore be4. In addition, the threshold cycle(Ct) values are transformed to quantities by using the comparative Ctmethod. For ge Norm, Ct values are transformed to relative non-normalized quantities(Q) according to the ge Norm manual(http://medgen.ugent.be/genorm/ge Norm_manual.pdf). The specific amplification efficiency was calculated using Excel 2007 software. The equation can then be expressed as: Q=E-△Ct, where E is the exponential amplification and △Ct=Sample Ct –min Ct, where sample Ct is the Ct value of the sample being transformed and min Ct is the lowest Ct value of each gene. Here, the highest relative quantities for each gene are set to 1 by using the 2-△Ctmethods. The gene expression is analyzed using the normalization factor calculated by the reference genes Ct values.【Results】This analysis of the candidate gene amplification efficiency found that six reference genetic standard curve slopes were close to-3.34. The correlation coefficient R2 has a range from 0.984 to 1.000. The efficiency of amplification reaction range is from94.77% to 108.78%. All indexes of the candidate reference genes were up to the requirements for the experiments performed. The stability of the six candidate reference genes ranked from high to low wereVv EF1 r =Vv EF1- α > Vv GAPDH >Vv ACTIN> Vv ACT1>Vv UBQ in the leaf and peel of the 8 table grapes. Vv EF1-α and Vv EF1 r were the most stabile genes of all six candidate reference genes in the leaf and peel respectively. And the V2/3value of the pairwise variances analysis is 0.104. The number of suitable reference genes was 2. So Vv EF1-α and Vv EF1 r were the most suitable reference genes group. The normalization factor was calculated on Vv EF1-α and Vv EF1 r by using ge Norm to analyze the target gene expression pattern. The gene expression of HSP70 in the leaf of the eight grape cultivars was generally higher than in the peel. The expression of HSP70 displayed the highest in‘Shenyu'and the lowest in‘Xi-ahei'within the leaves of the eight grape cultivars. And the expression of HSP70 displayed the highest in‘Jumeigui'and the lowest in‘Hupei 1#'within the peels of the eight grape cultivars.【Conclusion】According to the different varieties, the reference genes were variously expressed. The selection of stable reference genes was the key to the quantitative analysis of the gene expression. This study attempted to validate a set of candidate reference genes within the grapes for q RT-PCR normalization. Analysis of the expression stability using ge Norm revealed that Vv EF1-α and Vv EF1 r could be considered as appropriate reference genes in the leaves and peels of theeight grape cultivars based on the experiments performed.Whereas, other reference genes, Vv GAPDH, Vv UBQ, Vv ACT1 and Vv ACTIN, showed relatively low expression stability in the leaves and peels of the eight grape cultivars. Suitable reference genes should be selected on the basis of specific requirements, different experimental conditions, and the characteristics of the target genes using widely acceptable practical applications.: Grape; ge Norm; Reference genes; Normalization factor; Real-time quantitative PCR