Soltani Najafabadi Masood, Amirbakhtiar Nazanin
National Plant Genebank, Seed and Plant Improvement Institute, Agricultural Research, Education, and Extension Organization, Karaj, Iran.
Iran J Biotechnol. 2023 Apr 1;21(2):e3357. doi: 10.30498/ijb.2023.338375.3357. eCollection 2023 Apr.
Q-PCR is the method of choice for PCR- based transcriptomics and validating microarray-based and RNA-seq results. Proper application of this technology requires proper normalization to correct as much as possible errors propagating during RNA extraction and cDNA synthesis.
The investigation was performed to find stable reference genes in sunflower under shifting in ambient temperature.
Sequences of five well-known reference genes of Arabidopsis (, , , , and ) and one well-known reference gene inhuman, , were subjected to BLASTX against sunflower databases and the relevant genes were subjected to primer designing for q-PCR. Two sunflower inbred lines were cultivated at two dates so that anthesis occurred at nearly 30 °C and 40 °C (heat stress). The experiment was repeated for two years. Q-PCR was run on samples taken for two planting date separately at the beginning of anthesis for each genotype from leaf, taproots, receptacle base, immature and mature disc flowers and on pooled samples comprising of the tissues for each genotype, planting dates and also all tissues for both genotypes and both planting dates. Basic statistical properties of each candidate gene across all the samples were calculated. Furthermore, gene expression stability analysis was done for six candidate reference genes on Cq mean of two years using three independent algorithms, geNorm, Bestkeeper, and Refinder.
Designed primers for , , , , , and yielded a single peak in melting curve analysis indicating specificity of the PCR reaction. Basic statistical analysis showed that and had the highest and lowest expression levels across all the samples, respectively. appeared to be the most stable reference gene across all the samples based on the three used algorithms. Pairwise variation analysis revealed that for samples taken under ambient temperature of 30 °C, , , and for those taken under ambient temperature of 40 °C, , , and have to be used for normalization in q-PCR studies. Moreover, it is suggested that normalization to be based on , and for vegetative tissues and , , and Importin for reproductive tissues.
In the present research, proper reference genes for normalization of gene expression studies under heat stress conditions were introduced. Moreover, the presence of genotype-by- planting date interaction effects and tissue specific gene expression pattern on the behavior of the most three stable reference genes was indicated.
定量聚合酶链反应（Q-PCR）是基于聚合酶链反应的转录组学以及验证基于微阵列和RNA测序结果的首选方法。正确应用该技术需要进行适当的标准化，以尽可能校正RNA提取和cDNA合成过程中传播的误差。
本研究旨在寻找环境温度变化下向日葵中稳定的内参基因。
将拟南芥五个著名内参基因（，，，，和）以及人类一个著名内参基因的序列与向日葵数据库进行BLASTX比对，并对相关基因进行q-PCR引物设计。两个向日葵自交系在两个日期种植，使花期分别处于近30℃和40℃（热胁迫）。该实验重复进行两年。在每个基因型花期开始时，分别从叶片、主根、花托基部、未成熟和成熟盘花中采集样本进行Q-PCR，并对每个基因型、种植日期以及两个基因型和两个种植日期的所有组织的混合样本进行Q-PCR。计算每个候选基因在所有样本中的基本统计特性。此外，使用geNorm、Bestkeeper和Refinder三种独立算法，对六个候选内参基因两年的Cq均值进行基因表达稳定性分析。
为，，，，，和设计的引物在熔解曲线分析中产生单峰，表明PCR反应具有特异性。基本统计分析表明，在所有样本中表达水平最高，表达水平最低。基于所使用的三种算法，似乎是所有样本中最稳定的内参基因。成对变异分析表明，对于在30℃环境温度下采集的样本，，，对于在40℃环境温度下采集的样本，，，可用于q-PCR研究的标准化。此外，建议营养组织基于，，进行标准化，生殖组织基于，，和输入蛋白进行标准化。
在本研究中，引入了热胁迫条件下基因表达研究标准化的合适内参基因。此外，还表明了基因型与种植日期的交互作用以及组织特异性基因表达模式对最稳定的三个内参基因行为的影响。
