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

Chapters

  • 00:04Title
  • 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

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

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