Abstract
Small RNAs mediate posttranscriptional gene silencing in plants and animals. This often occurs in specific cell or tissue types and can be necessary for their differentiation. Determining small RNA (sRNA) localization patterns at cellular resolution can therefore provide information on the corresponding gene regulatory processes they are involved in. Recent improvements with in situ hybridization methods have allowed them to be applied to sRNAs. Here we describe an in situ hybridization protocol to detect sRNAs from sections of early staged Arabidopsis thaliana (Arabidopsis) embryos.
Highlights
RNA in situ hybridization is a technique that utilizes antisense oligonucleotide probes to detect complementary RNAs in a tissue of interest. This enables the characterization of RNA localization patterns and yields insights into their functions
The length of the probe required for stable target RNA duplex formation can be reduced facilitating the detection of small RNAs from various species and tissue types [1–8]
Once probes are designed and prepared, the experiment takes 7 days to complete: tissue fixation and dehydration, clearing, embedding and sectioning, proteinase K digestion, EDC fixation and probe hybridization, washing and antibody reaction, and colorimetric reaction and mounting. This protocol was optimized for sectioned Arabidopsis embryos, it can be adapted to other tissue types with modifications as noted
Summary
RNA in situ hybridization is a technique that utilizes antisense oligonucleotide probes to detect complementary RNAs in a tissue of interest. This enables the characterization of RNA localization patterns and yields insights into their functions. An additional fixation step uses 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to immobilize the 50 monophosphates of sRNAs to the specimen’s protein matrix. This enhanced the sensitivity and robustness of sRNA in situ hybridization methods [9, 10]. The original version of this chapter was revised.
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