In plant cells, microRNA (miRNA) precursors formed by transcription first fold into a stem-loop structure then shear to form mature miRNAs under the action of DCL1. In different plant tissues and organs and at different developmental periods, miRNA expression profiles and regulation are different. The miRNA detection service provided by Lifeasible can be applied to the study of miRNA biological functions in a wide range of plant species.

Parallel analysis of RNA end (PARE)

We used the PARE technique to enrich all poly(A)-containing mRNAs with an endonuclease recognition site on the RNA junction. The spliced mRNA is reverse transcribed into cDNA, which is enzymatically cleaved, and a DNA splice is added. We designed primers based on the sequences of both junctions, amplified by PCR, and cloned into a library for deep sequencing. The sequences obtained after sequencing were subjected to sequence comparison with the original mRNA transcripts by bioinformatics. The combination of high-throughput sequencing and PARE can be used to study the tissue-specific expression of miRNAs with target genes.

miRNA overexpression

Driving the transcription of pre-miRNAs with strong promoters is currently the most widely used method in miRNA overexpression systems. We genetically overexpressed individual miRNAs to obtain transgenic plants with enhanced or reduced tolerance to biotic or abiotic stresses.

Construction of Nicotiana tabacum transient expression system

Constructing the Nicotiana tabacum transient expression system to observe the expression strength of reporter gene proteins can get more reliable and graphic experimental evidence. We used green fluorescent protein (GFP) and β-glucuronidase (GUS) as indicator markers to detect the degradation of target genes by miRNA.

• Sequence fragments of target genes of miRNAs fused with reporter genes were constructed into plant expression vectors.
• The constructed cloning vector and miRNA were introduced into Nicotiana tabacum at the same time, and the reporter gene was transcribed and expressed under promoter drive.
• If the miRNA can act on the target gene sequence, the transcript will be cleaved and degraded, and the expression of the reporter gene will not be detected or reduced. Conversely, if the miRNA cannot act on the target gene sequence, expression of the reporter gene will be detected.

Simultaneous detection of miRNAs as well as mRNAs at the single-cell level in plant tissues

Simultaneous detection of miRNAs as well as mRNAs in specific tissues can help us to predict regulatory relationships between miRNAs and mRNAs. We achieved simultaneous detection of miRNAs and mRNAs by using sequence-specific probes, which enhance the detection signals by rolling amplification and achieve high signal-to-noise ratios at the single-cell level of application. Based on this approach, we have successfully applied and demonstrated the dynamic expression of miRNAs and mRNAs in our projects in rice, maize, and other plants.

Lifeasible offers methods to study how miRNAs regulate plant tissue development; for more information, please feel free to contact us.

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