| Northern Blot of tRNA in Yeast
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Author:
Date:
2013-04-05
[Abstract] tRNAs are small RNAs around 70-90 nt. tRNAs are different from many other small RNAs in that they are very abundant, which makes it difficult to study their transcriptional regulation by traditional northern blot. Traditional Northern blot involves incorporation of radioactive nucleotides through polymerization, however, tRNA is too short for polymerization. Traditional Northern blot detects changes in RNA levels, however, tRNA are so abundant that small changes in their levels will escape detection. For these reasons, metabolic labeling by radioactive Uracil has been used instead. However, metabolic labeling can only examine changes in total tRNA, but cannot distinguish different types of tRNAs. The following protocol describes a method to examine individual tRNA gene transcription by ...
[摘要] tRNA是约70-90nt的小RNA。 tRNA与许多其他小RNA不同，因为它们是非常丰富的，这使得难以通过常规Northern印迹研究它们的转录调节。 传统的Northern印迹涉及通过聚合掺入放射性核苷酸，然而，tRNA对于聚合来说太短。 传统的Northern印迹检测RNA水平的变化，然而，tRNA是如此丰富，其水平的小变化将逃脱检测。 由于这些原因，已经使用了放射性尿嘧啶的代谢标记。 然而，代谢标记只能检查总tRNA的变化，但不能区分不同类型的tRNA。 以下方案描述了通过Northern印迹检查单个tRNA基因转录的方法。
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| Metabolic Labeling of Yeast RNA with Radioactive Uracil
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Author:
Date:
2013-04-05
[Abstract] To examine gene expression, Northern blot or Real-Time PCR can be used to detect low abundant RNA such as mRNA. However, for high abundant RNAs such as rRNA and tRNA, Northern blot will not be able to discriminate the newly synthesized RNA from total RNA. Therefore, metabolic labeling is necessary to evaluate the expression of rRNA and tRNA genes. In this protocol, I describe a step-by-step method for labeling yeast RNA with radioactive uracil and examine the synthesis of these high abundant RNAs.
[摘要] 为了检查基因表达，可以使用Northern印迹或实时PCR来检测低丰度RNA如mRNA。 然而，对于高丰度的RNA如rRNA和tRNA，Northern印迹将不能区分新合成的RNA与总RNA。 因此，代谢标记是必要的，以评估rRNA和tRNA基因的表达。 在这个协议，我描述了一个分步的方法，用放射性尿嘧啶标记酵母RNA，并检查这些高丰度RNA的合成。
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