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. 2008 Oct 20:1:93.
doi: 10.1186/1756-0500-1-93.

DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques

Luis Oñate-Sánchez  1 , Jesús Vicente-Carbajosa
Affiliations

DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques

Luis Oñate-Sánchez et al. BMC Res Notes. .

Abstract

Background: High throughput applications of the reverse transcriptase quantitative PCR (RT-qPCR) for quantification of gene expression demand straightforward procedures to isolate and analyze a considerable number of DNA-free RNA samples. Published protocols are labour intensive, use toxic organic chemicals and need a DNase digestion once pure RNAs have been isolated. In addition, for some tissues, the amount of starting material may be limiting. The convenience of commercial kits is often prohibitive when handling large number of samples.

Findings: We have established protocols to isolate DNA-free RNA from Arabidopsis thaliana tissues ready for RT-qPCR applications. Simple non-toxic buffers were used for RNA isolation from Arabidopsis tissues with the exception of seeds and siliques, which required the use of organic extractions. The protocols were designed to minimize the number of steps, labour time and the amount of starting tissue to as little as 10-20 mg without affecting RNA quality. In both protocols genomic DNA (gDNA) can be efficiently removed from RNA samples before the final alcohol precipitation step, saving extra purification steps before cDNA synthesis. The expression kinetics of previously characterized genes confirmed the robustness of the procedures.

Conclusion: Here, we present two protocols to isolate DNA-free RNA from Arabidopsis tissues ready for RT-qPCR applications that significantly improve existing ones by reducing labour time and the use of organic extractions. Accessibility to these protocols is ensured by its simplicity and the low cost of the materials used.

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Figures

Figure 1
Figure 1
DNA-free RNA isolation protocols. Several steps common for both protocols are summarized at the top of the figure. General considerations taken when working with RNA, such as glassware sterilization or wearing gloves, are also applicable to these protocols.
Figure 2
Figure 2
PCR to detect gDNA, RNA electropherograms and RT-qPCR on Arabidopsis seeds and leaves. (A) PCR products after 35 cycles with β-tubulin specific oligonucleotides on DNase treated total RNA isolated from dry (0), stratified (S) and germinating seeds (6 to 48 hours); (-): no template; (+): gDNA; (M): molecular marker. (B) qPCR with AtDOF31 specific oligos on cDNAs prepared from total RNA samples isolated from leaves of wild-type (Col-0) or transgenic plants overexpressing the AtDOF31 gene (lines 1 to 6). Expression levels relative to ubiquitin (UBQ; At5g25760) are shown and numbers in italic inside the graph indicates fold differences with Col-0. (C) Electropherograms of 500 ng total RNA samples from seeds and siliques (Agilent 2100 Bioanalyzer). Electrophoretic RNA migration time and fluorescence values correlate with RNA size and quantity, respectively, and are calculated by comparison to a RNA ladder (Agilent Technologies, Palo Alto, CA, USA).
Figure 3
Figure 3
qPCR on selected genes to determine protocol 2 specificity and sensitivity. qPCR with gene (indicated inside the graph) specific oligonucleotides on cDNAs prepared from total RNAs described in Figure 1A. Expression levels relative to ubiquitin (UBQ; At5g25760) are shown.
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