Variation of Expression Levels of Seven Housekeeping Genes at Different Life-History Stages in Porphyra yezoensis

In order to identify the optimal internal control for relative real-time PCR when studying target gene expression in the red alga Porphyra yezoensis, we quantified the expression of seven housekeeping genes (18S ribosomal RNA, 30S ribosomal protein S8, Polyubiquitin-2, Glyceraldehyde-3-phosphate dehydrogenase, Elongation factor 1-alpha, Beta-tubulin and Actin 3) at different life-history stages. Absolute quantification was done by normalization to total RNA quantity and by normalization to genomic DNA quantity. We used these two normalization approaches, comparing the differences of expression levels of all candidate housekeeping genes between any two generations and across three life-history stages (filamentous sporophytes, leafy gametophytes and conchospores). We found GAPDH had the best stability in all cases and we recommend that GAPDH be considered as a potential internal control for gene expression studies at different life-history stages in P. yezoensis.     

Epicocconone staining: A powerful loading control for Western blots

Western blot analysis is routinely employed for quantifying differences in protein levels between samples. To control equal loading and to arithmetically compensate loading differences, immunodetection of housekeeping proteins is commonly used. Due to potential biases, this approach has been criticized. Here, we evaluate epicocconone‐based total protein staining (E‐ToPS) as an alternative. We compared it with two other total protein stainings (Coomassie and Sypro Ruby) and with immunodetection of housekeeping proteins (β‐tubulin and glyceraldehyde 3‐phosphate dehydrogenase). Evaluation comprised both the natural and the synthetic epicocconone compound. Both compounds produced highly congruent results and showed more sensitive (≤ 1 μg) and less variable staining properties than the other variants. The high sensitivity of E‐ToPS, covering minute protein amounts, makes it a powerful loading control, especially for precious samples. Regarding biological and technical variances, E‐ToPS outperformed immunostaining against β‐tubulin and glyceraldehyde 3‐phosphate dehydrogenase. Furthermore, E‐ToPS had no impact on subsequent immunodetection, allowing for an early control of proper loading prior to immunodetection. In contrast to earlier studies, we found logarithmic staining properties for E‐ToPS, which should be considered when using it for arithmetic normalization. In conclusion, we demonstrate the superior power of E‐ToPS as a loading control for Western blots.     

DNA芯片技术中利用内标对数据归一化后检测基因的表达变化

在DNA芯片技术中 ,通过反转录反应 ,由mRNA合成带有荧光标记物的cDNA的过程中 ,往往要参入已知质量的poly(A) + RNA ,以对DNA芯片的检测灵敏度进行归一化处理 .通过体外转录的方法 ,以真核生物的cDNA克隆中的DNA片段为模板合成poly(A) +RNA ,对之定量后 ,以不同的质量比参入到样品的反转录体系中 ,代表不同的RNA拷贝丰度 ,从而对DNA芯片检测的灵敏度进行了定量 ,并得到DNA芯片上杂交点的荧光信号强度与基因表达的RNA拷贝数成正相关的关系 .利用含有内标的DNA芯片检测了热击反应后酵母细胞的基因表达变化 ,结果与Northern印迹方法检测结果是相符的     

Stain-Free total protein staining is a superior loading control to β-actin for Western blots

Semi-quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, Ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain-Free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain-Free staining as an alternative to β-actin or the protein stain Ponceau S showed that Stain-Free staining was superior to β-actin and as good as or better than Ponceau S staining as a loading control for Western blots.