A plastome primer set for comprehensive quantitative real time RT-PCR analysis of Zea mays: a starter primer set for other Poaceae species

Richard M Sharpe,Sade N Dunn,A Bruce Cahoon

doi:10.1186/1746-4811-4-14

Abstract

BackgroundQuantitative Real Time RT-PCR (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RT)PCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species.ResultsPrimers were designed and successfully optimized for 57 of the 104 reported genes in the maize plastome plus two nuclear genes. All 59 primer pairs produced single amplicons after end-point reverse transcriptase polymerase chain reactions (RT-PCR) as visualized on agarose gels and subsequently verified by q2(RT)PCR. Primer pairs were divided into several categories based on the optimization requirements or the uniqueness of the target gene. An in silico test suggested the majority of the primer sets should work with other members of the Poaceae family. An in vitro test of the primer set on two unsequenced species (Panicum virgatum and Miscanthus sinensis) supported this assumption by successfully producing single amplicons for each primer pair.ConclusionDue to the highly conserved chloroplast genome in plant families it is possible to utilize primer pairs designed against one genomic sequence to detect the presence and abundance of plastid genes or transcripts from genomes that have yet to be sequenced. Analysis of steady state transcription of vital system genes is a necessary requirement to comprehensively elucidate gene expression in any organism. The primer pairs reported in this paper were designed for q2(RT)PCR of maize chloroplast genes but should be useful for other members of the Poaceae family. Both in silico and in vitro data are presented to support this assumption.

Highlights

Quantitative Real Time reverse transcriptase polymerase chain reactions (RT-PCR) (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids
Plastids have maintained a small remnant genome with a species-specific number of genes mostly involved in photosynthesis and gene expression
In this paper we describe the development and empirical optimization of primer pairs to amplify each rRNA and mRNA from maize plastids

Summary

Introduction

Quantitative Real Time RT-PCR (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RT)PCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species. Chloroplasts are semi-autonomous organelles believed to have developed from free-living photosynthetic bacteria [1,2] They are members of a diverse and flexible family of organelles called plastids that are responsible for photosynthesis plus other essential biosynthetic pathways and cellular functions. Plastids have maintained a small remnant genome with a species-specific number of genes mostly involved in photosynthesis and gene expression. The full function and development of all the plastid types, requires thousands of nuclear encoded gene products.

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