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Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy
JoVE Journal
Genetica
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JoVE Journal Genetica
Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

DOI:
10.3791/60019-v

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07:26 min

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July 29, 2019

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, , , , , , ,
1State Key Laboratory of Conservation and Utilization of Subtropical Agro-Bioresources,South China Agricultural University, 2Guangdong Provincial Key Laboratory of Plant Molecular Breeding, College of Agriculture,South China Agricultural University

Capitoli

  • 00:04Titolo
  • 00:39Preparation of Crude Mitochondrion from Maize Developing Kernels
  • 01:45RNA 5’ Polyphosphatase Treatment and Circularization
  • 02:33Reverse Transcription and Normalization
  • 03:30PCR Amplification and Determination of Transcript Termini
  • 04:34Verification of the cRT-PCR Mapping Results by RNA Gel Blot Hybridization
  • 05:00Discrimination of Primary and Processed 5’ Ends
  • 05:26Determination of Maize mRNA Termini Using the Circular RT-PCR-based Strategy
  • 07:05Conclusion

Summary

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We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5' polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5' triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

Tags

MitochondrionMaizePrimary TranscriptsProcessed TranscriptsCircular RT-PCRRNA NormalizationFive Prime Polyphosphatase TreatmentRNA ExtractionReverse TranscriptionPCR

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