miScript Plant RT Kit
For cDNA synthesis of plant miRNAs and other 3' modified small RNAs
The miScript Plant RT Kit is an unbiased, universal cDNA synthesis kit for small RNAs with 3’-end, 2’-O-Me modifications. The kit has been designed and optimized for real-time PCR-based small RNA expression analysis together with miScript SYBR Green PCR Kit and miScript miRNA PCR Arrays or miScript Primer Assays. These functionally verified assays and arrays are available for rice, corn, soybean, tobacco, and other significant species in agriculture and plant research. Small RNAs with a 2′-O-Me modification on the 3′ terminal base, like plant mature miRNAs and piwi-interacting RNAs (piRNAs), are refractory to polyadenylation, and cannot be efficiently reverse transcribed using a polyadenylation-based cDNA synthesis approach. The specialized miScript Plant RT Kit overcomes this by first ligating an adaptor to the 3′ terminus of plant miRNAs. Unlike polyadenylation, this ligation reaction is not negatively inhibited by the presence of a 2′-O-Me modification on the 3′ terminal base of plant miRNAs. Reverse transcription can be performed following ligation.
The miScript Plant RT Kit, together with the components of the miScript Plant qPCR System, ensures sensitive, specific miRNA detection and quantification from plant samples. As many miRNAs are expressed at low levels, sensitivity is critical to ensure reliable data. The miScript Plant qPCR System provides exceptional sensitivity, a broad dynamic range, and consistently high amplification efficiencies. The high performance of the miScript Plant qPCR System ensures highly reproducible results between technical and biological replicates on the first attempt.
Mature miRNAs are naturally occurring, 22-nucleotide, noncoding RNAs that mediate post-transcriptional gene regulation. Plant miRNAs and piRNAs possess a 2′-O-Me residue on their 3′ terminal base. Due to this, ligation-mediated reverse-transcription is required for the universal cDNA synthesis of plant miRNAs and piRNAs.
The miScript Plant RT Kit includes all of the reagents required to perform universal, ligation-mediated reverse transcription of plant mature miRNAs, including 10x miScript Ligation Buffer, 10x miScript Plant Adaptor, 2x miScript Ligation Activator, miScript Plant RNA Ligase, 10x miScript Plant RT Nucleics, 5x miScript Plant RT Buffer, and miScript Plant Reverse Transcriptase. When ligation-mediated reverse transcription is performed, small RNAs are selectively converted into cDNA. In the first step of the reaction, an adaptor is ligated to the mature miRNAs. In the second step of the reaction, the ligated miRNAs are reverse-transcribed into cDNA.
10x miScript Plant Adaptor contains an optimized pre-adenylated adaptor and a synthetic RNA control (miScript Plant Ligation Control [miPLC]) that is used to assess ligation-mediated reverse-transcription performance. 10x miScript Plant RT Nucleics contains dNTPs, RT primers, and an internal synthetic RNA-DNA hybrid control (miScript Plant Reverse-Transcription Control [miPRC]) that is used to test reverse transcription performance. The unique, patent-pending formulation of 5x miScript Plant RT Buffer facilitates the selective conversion of mature miRNAs into cDNA, enabling miRNA quantification with either miScript miRNA PCR Arrays or miScript Primer Assays.
Real-time PCR miRNA expression profiling
cDNA prepared using the miScript Plant RT Kit serves as the template for real-time PCR analysis using a plant miScript miRNA PCR Array (which contains miRNA-specific miScript Primer Assays) and the miScript SYBR Green Kit, which contains the miScript Universal Primer (reverse primer) and QuantiTect SYBR Green PCR Master Mix. To profile mature miRNA expression, a premix of cDNA, miScript Universal Primer, QuantiTect SYBR Green PCR Master Mix, and RNase-free water is added to the miScript miRNA PCR Array.
Real-time PCR miRNA expression quantification
cDNA prepared using the miScript Plant RT Kit serves as the template for real-time PCR analysis using a plant miScript Primer Assay and the miScript SYBR Green Kit. To quantify mature miRNA expression, a mix of cDNA, miScript Primer Assay, miScript Universal Primer, QuantiTect SYBR Green PCR Master Mix, and RNase-free water is prepared and added to PCR tubes, Rotor-Discs, or plates.
Reverse transcription reactions are easy to perform. Total RNA containing 3′-end, 2′-O-Me modified small RNAs is used as starting material for ligation-mediated reverse transcription reactions. Reverse transcription is a straightforward procedure which includes incubation of the reaction at 37°C for 1 hour, followed by inactivation of the reaction by briefly incubating at 95°C.
The miScript Plant RT Kit is used as part of the miScript Plant PCR System for:
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