A method for extracting high‐quality RNA from diverse plants for next‐generation sequencing and gene expression analyses

Abstract

• Premise of the study: To study gene expression in plants, high-quality RNA must be extracted in quantities sufficient for subsequent cDNA library construction. Field-based collections are often limited in quantity and quality of tissue and are typically preserved in RNAlater. Obtaining sufficient and high-quality yield from variously preserved samples is essential to studies of comparative biology. We present a protocol for the extraction of high-quality RNA from even the most recalcitrant plant tissues.• Methods and Results: Tissues from mosses, cycads, and angiosperm floral organs and leaves were preserved in RNAlater or frozen fresh at −80°C. Extractions were performed and quality was measured for yield and purity.• Conclusions: This protocol results in the extraction of high-quality RNA from a variety of plant tissues representing vascular and nonvascular plants. RNA was used for cDNA synthesis to generate libraries for next-generation sequencing and for expression studies using quantitative PCR (qPCR) and semiquantitative reverse transcription PCR (RT-PCR).

Practical considerations for plant phylogenomics

“…A number of phylotranscriptomic protocols have been developed in various plant lineages (Johnson et al, 2012;Yockteng et al, 2013;Jordon-Thaden et al, 2015;Yang et al, 2017). Suitable extraction protocols can be lineage specific, and we recommend starting from existing protocols that have proven effective in closely related plant groups.…”

Boosting photosynthetic machinery and defense priming with chitosan application on tomato plants infected withFusarium oxysporumf. sp.lycopersici

“…were first treated with chitosan, and 24 h after this treatment, the same plants were infected with Fol59, then after 24 h of Fol59 inoculation (forty-eight hours after chitosan treatment), the foliar part of the plants was collected and flash frozen in liquied nitrogen. Subsequently, total RNA was extracted using the protocol by (Yockteng et al, 2013). The following treatments were assessed: (i) Control (plants treated with water), (ii)…”

An efficient field and laboratory workflow for plant phylotranscriptomic projects1

“…We tested five alternative RNA extraction protocols. These include TRIzol option 1 from Jordon-Thaden et al (2015), the Aurum TM Total RNA Mini Kit (BIO-RAD) following the manufacturer’s protocol, the QIAGEN RNeasy Mini Kit (QIAGEN) following the manufacturer’s protocol, the PureLink protocol (Ambion; see protocol in Appendix 3; Yockteng et al, 2013) and the hot acid phenol-LiCl-RNeasy Mini Kit protocol [see protocol in Appendix 4, modified from Protocol 12 of Johnson et al (2012)]. We had approximately 10-30% success rate with BIO-RAD, QiaGen and TRIzol protocols, whereas the PureLink kit had close to 100% success rate and only failed when the sample itself was degraded or highly mucilaginous.…”

“…Previous literature on phylotranscriptomic methods has focused on RNA extraction (Johnson et al, 2012; Yockteng et al, 2013; Jordon-Thaden et al, 2015) and data analyses (Yang and Smith, 2013; Yang and Smith, 2014). However, as phylotranscriptomic studies expand to non-model systems that often require field sampling, the logistics of obtaining fresh tissues becomes the limiting factor.…”