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High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

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An efficient full-length cDNA amplification strategy based on bioinformatics technology and multiplexed PCR methods.

Nan Chen¹, Wei-Min Wang¹, Huan-Ling Wang^1,2

¹Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, 430070, Wuhan, PR China.

Scientific Reports

|January 14, 2016

Summary

This study introduces a novel bioinformatics and multiplexed PCR strategy to amplify full-length cDNA and promoter sequences efficiently. The method enables rapid isolation of valuable genetic information for researchers.

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Area of Science:

Molecular Biology
Genomics
Bioinformatics

Background:

Isolating full-length cDNA and promoter sequences is crucial for understanding gene regulation.
Traditional methods can be time-consuming, expensive, and inefficient, especially for limited samples.

Purpose of the Study:

To develop a novel, efficient, and cost-effective strategy for amplifying full-length cDNA and promoter sequences.
To combine bioinformatics with multiplexed PCR techniques for enhanced sequence amplification.

Main Methods:

Modified 3' RACE with a novel oligo(dT)-anchor primer for 3' cDNA end amplification.
Multi-round TAIL-PCR or touch-down PCR with arbitrary degenerate (AD) and sequence-specific reverse (SPR) primers for 5' cDNA end amplification.
In silico analysis of assembled fragments against reference genomes and cis-element screening for promoter identification and validation.

Main Results:

Successful amplification of full-length cDNA and promoter sequences using the developed strategy.
Demonstrated efficiency and speed in isolating target sequences.
Validated putative 5' ends and promoter regions through in silico and experimental methods.

Conclusions:

The novel strategy offers an operable, inexpensive, efficient, and speedy solution for researchers needing to isolate limited full-length cDNA sequences.
This approach enhances the ability to study gene structure and regulation from limited biological material.