Abstract
microRNAs are a class of non-coding small RNAs (sRNAs) that are important regulators of gene expression at the post-transcriptional level by mRNA cleavage or translation inhibition. Another class of sRNAs are siRNAs, which also regulate gene expression but by causing DNA methylation. This epigenetic regulatory role has been observed for some miRNAs as well. The use of sRNAs allows the development of biotechnological applications in plants. To develop these types of applications, and to better understand the natural roles they play, it is important to be able to detect and to localize these sRNAs at the plant tissue level. Sometimes, in crop plants this can be challenging. Therefore, we developed a tissue printing hybridization protocol for easy and efficient detection of sRNAs and demonstrate this by the analysis of the spatio-temporal expression patterns of the miRNAs miR159 and miR164 in fruits of various crop plants. Moreover, we show the possibility to also detect the expression of miRNAs in fruit juice using a dot blot hybridization approach.
Highlights
MicroRNAs are a class of small RNAs that are fundamental regulatory elements of eukaryotic genomes (Voinnet, 2009) and their widespread conservation and divergence in the plant kingdom has been demonstrated (Chavez Montes et al, 2014)
In this work we aimed to develop an easy protocol that would allow the in situ detection of miRNAs in relatively large plant tissues or organs
For this we used the combination of tissue printing and hybridization
Summary
MicroRNAs (miRNAs) are a class of small RNAs (sRNAs) that are fundamental regulatory elements of eukaryotic genomes (Voinnet, 2009) and their widespread conservation and divergence in the plant kingdom has been demonstrated (Chavez Montes et al, 2014). Detection of the spatio-temporal expression of miRNAs is critical to understand their function (van Rooij, 2011) Several techniques such as Northern-blot hybridization (Pall and Hamilton, 2008), microarray analysis (Yin et al, 2008), stem-loop RT-PCR analysis (Chen et al, 2005), or sRNA-seq analysis (Chavez Montes et al, 2014) permit the identification of miRNA expression patterns, which may suggest their involvement in certain biological processes. Tissue printing has been defined as ‘the art and science of visualizing cellular material and information that are transferred to a receptive surface when the cut surfaces of section of tissues or organs are pressed against such a surface’ (Varner and Ye, 1994) This technique does not require RNA extraction or the preparation of thin tissue sections, and it allows the simultaneous analysis of many samples. To date almost no examples have been reported for the detection of miRNAs in plants using tissue printing (e.g., Rosas-Cárdenas et al, 2015)
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