圖1. 面對全面轉錄變化的挑戰，一項新研究引進了一種，使用人工摻入技術，來提高植物基因活性分析的準確性。此方法提供了有關植物如何對，諸如溫度等環境刺激作出反應之更清晰的洞察力。因此，可望在植物科學研究中，廣泛被採用。
A new study introduces a technique using artificial spike-ins to improve the accuracy of gene activity analysis in plants, addressing the challenge of global transcription changes. This method provides clearer insights into how plants respond to environmental stimuli, such as temperature, and is hoped to be widely adopted in plant science research.
Researchers have published a simple trick that improves the accuracy of techniques that help us understand how external variables – such as temperature – affect gene activity in plants.
研究人員們已經發表一種，改善協助我們瞭解，諸如溫度等外部變數，如何影響植物中，基因活性之技術準確性的簡單技巧。
“There are really two contributions here,” says Colleen Doherty, corresponding author of a paper on the work and an associate professor of molecular and structural biochemistry at North Carolina State University. “First, we’re raising the visibility of a problem that many of us in the plant research community were unfamiliar with, as well as highlighting the solution. Second, we’ve demonstrated that addressing this problem can make a significant difference in our understanding of gene activity in plants.”
該項研究論文的通訊撰文人，美國北卡羅萊納州立大學分子暨結構生物化學副教授，Colleen Doherty宣稱：「在這點上，確實有兩項貢獻。第一項，除了強調該解決方案之外，我們正在提升，於植物研究界中，諸多人不熟悉之問題的能見度。第二項，在瞭解植物中基因活性上，我們已經證實，面對此問題能產生重大的差異。」
At issue is a technique called RNA-seq analysis, which is used to measure changes in gene activity – i.e., when genes are actively transcribing to produce proteins.
爭論的是一種，被用於測量基因何時積極轉錄，以產生蛋白質之活性變化，被稱為RNA-seq分析的技術。
“We use RNA-seq analysis to assess how plants respond to various stimuli, or changes in their environment,” Doherty says. “It’s used widely because it’s a relatively easy and inexpensive way to monitor plant responses.”
Doherty宣稱：「我們使用RNA-seq分析，來評估植物如何，對各種刺激或於其環境中的變化，作出反應。這被廣泛使用，因為它是一種，相對上簡單且廉價之監測植物反應的方法。」
For example, researchers can use RNA-seq analysis to see which genes are turned on when a plant is experiencing drought conditions, which then informs the development of new plant varieties that are drought resistant.
譬如，研究人員們能使用RNA-seq分析，來瞭解當植物經歷乾旱狀況時，哪些基因被開啟，之後為開發抗旱的新植物品種，提供資訊。
But there’s a specific challenge related to RNA-seq analysis, which Doherty and her collaborators ran into by accident.
不過，Doherty及其合作者們偶然碰上一種，與RNA-seq分析相關的特殊挑戰。
“We were monitoring how plants respond to different temperatures at multiple times of day, and the results we got were wildly divergent,” Doherty says. “We initially thought we might be doing something wrong. But when we began looking into it, we learned that animals and yeasts are known to have global changes in transcription based on variables such as the time of day or nitrogen deprivation.”
Doherty宣稱：「我們在一天中的多個時間，監測植物如何對不同溫度作出反應。結果，我們得到的此些結果，是極分岐的。起初我們認為，或許出了什麼差錯。不過，當我們開始探究時，我們得悉，在根據諸如一天或氮剝奪之時間，等變數的轉錄中，動物及酵母菌已知經歷整體變化。」
In other words, researchers want to see how specific variables – such as increased temperature – affect transcription in specific genes. But there are some variables – like time of day – that can increase or decrease transcription in all the genes. This can throw off researchers’ ability to draw conclusions about the specific variables they want to study.
換句話說，研究人員們想瞭解，特定變數(諸如升高的溫度)如何影響，於特定基因中的轉錄。不過，於所有此些基因中有一些，像一天中的時間般，能增加或減少轉錄的變數。這會消除研究人員們，有關他們想要研究之特定變數，得出結論的能力。
“Luckily, we found that this problem is sufficiently well-established among researchers who work on non-plant species that they have developed a method to account for it, called an artificial spike-in,” Doherty says. “These and similar techniques have been used in plant science in other contexts and when using older techniques and technologies. But for whatever reason, our field didn’t incorporate artificial spike-ins into our methodology when we adopted RNA-seq analysis.”
Doherty宣稱：「幸運的是，我們發現，此問題在針對非植物物種進行研究的人員中，是充分得到證實的。他們已經開發出一種，被稱為人工插入的方法，來解釋此問題。這些及類似的技術，在其他背景中及使用較老舊的技術與科技時，已經被用於植物科學。不過，無論出於何種原因，當我們採用RNA-seq分析時，我們的領域並沒有將人工插入納入我們的方法論中。」
Artificial spike-ins make use of pieces of foreign RNA that are unlike anything in the plant’s genome, meaning that the foreign RNA will not be confused with anything the plant itself produces.
人工插入利用了，與植物基因體中任何東西不同的外來RNA片段。這意味著，外來RNA不會與植物本身產生的任何東西被混淆。
Researchers introduce the foreign RNA into the analysis process at the beginning of the experiment. Because global changes in transcription will not affect the foreign RNA, it can be used as a fixed benchmark that allows researchers to determine the extent to which there is an overall increase or decrease in RNA that the plant itself is producing.
於實驗開始時，研究人員們將外來的RNA引入此分析過程中。由於，在轉錄中的整體變化，不會影響外來的RNA。因此，它能被使用作為一種，使研究人員們得以決定，於植物本身產生之RNA，有總體增加或減少的程度。
“When we used artificial spike-ins to account for global changes in transcription, we found that the differences in plants exposed to temperature changes at different times of day were actually even greater than we anticipated,” Doherty says.
Doherty宣稱：「當我們使用了人工插入，來解釋轉錄的整體變化時。我們發現，在一天中的不同時間，被暴露於溫度變化的植物中，差異實際上比我們預期的更大。」
“The artificial spike-in gave us more accurate information and greater insight into how plants are behaving at night – since we found that global transcription was higher at night. Before we adopted the use of artificial spike-ins, we were missing a lot of what was happening at night.
人工插入賦予了我們更準確的資訊，更深入洞察植物於夜間如何運作。因為我們發現，整體轉錄於夜間較高。在我們採用人工插入之前，我們錯過了很多，於夜間發生的事。
“Artificial spike-ins are an elegant solution to a challenge many of us in the plant research community didn’t even know was there,” Doherty says. “We’re optimistic this technique will improve the accuracy of transcriptional analysis in the wide variety of conditions that can affect global transcription in plant species. And that, in turn, may help our research community garner new insights into the species we study. We didn’t develop this solution – artificial spike-ins – but we really hope it garners more widespread use in plant science.”
Doherty宣稱：「人工插入是一種，在我們植物研究界中，諸多人甚至不知道之挑戰的極佳解決方案。我們是樂觀的，該項技術將改善，在會於植物物種中，影響整體轉錄之各種條件下，轉錄分析的準確性。而且，依序可能有助於我們研究界，獲得我們研究之物種的新洞察力。我們沒有展開此解決方案(人工插入)，不過我們真的期盼，它在植物科學中，獲到更普及的應用。」
網址：https://scitechdaily.com/how-a-simple-trick-is-transforming-plant-gene-analysis/
翻譯：許東榮
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