RNA Extraction from Plant Tissue with Homemade Acid Guanidinium Thiocyanate Phenol Chloroform (AGPC)

Abstract

Gene expression studies are a powerful technique to study biological processes, and isolating RNA that is pure, intact, and in sufficient amounts for downstream applications is key. Over the years, the field has moved to the use of commercial kits and ready-made extraction buffers for RNA isolation. This became particularly problematic during the COVID-19 crisis when supply chains were affected and when RNA extraction and analysis reagents were suddenly scarce at a time when they were particularly required. Acid guanidinium thiocyanatephenol-chloroform (AGPC) is one of the oldest RNA extraction solutions, in use since 1987. It is known as a ready-made solution, sold under different brand names, and is typically the most expensive reagent in the RNA extraction process. In this article, we describe how to prepare a low-cost homemade AGPC solution and provide tips on how to use it for obtaining high-quality RNA, as well as describe possible modifications for different conditions. The protocol is based on a phase separation, where RNA is maintained in the aqueous phase and DNA and proteins remain in the interphase and organic phase. After cleaning, precipitation, and resuspension steps, the RNA is ready to be quantified and used for downstream applications. By following this protocol, good yields of high-quality RNA can be obtained from a wide variety of tissues and organisms, and we exemplify the approach here using plant tissues. Some plant tissues contain extra interferents (such as sugars), and for high-quality RNA isolation from those tissues, an alternate protocol is provided.

A comparative analysis of RNA isolation methods optimized for high-throughput detection of viral pathogens in California’s regulatory and disease management program for citrus propagative materials

“…Despite these low 260/230 ratio values, all three isolation methods produced RNA that was successfully used for the detection of all targeted graft-transmissible pathogens of citrus using various RT-PCR-based assays without any reaction inhibition (Table 2). Our comparative and regulatory testing results using over 6,000 samples are in agreement with multiple studies that have demonstrated that there is no correlation between 260/230 absorbance ratios of RNA extracts and performance of downstream RT-PCR or RT-qPCR analysis (Figure 2B) (Cicinnati et al, 2008;Ahlfen and Schlumpberger, 2010;Gallagher, 2017;Zepeda and Verdonk, 2022). Specifically for guanidine, it has been calculated that up to 100 mM in an RNA sample does not compromise RT-PCR reactions (Ahlfen and Schlumpberger, 2010).…”

“…Specifically for guanidine, it has been calculated that up to 100 mM in an RNA sample does not compromise RT-PCR reactions (Ahlfen and Schlumpberger, 2010). Therefore in the case of RNA isolation from citrus tissues using guanidine, the 260/230 absorption ratios should be used as a complimentary RNA purity measurement, and should never be used as a major determinant of a sample's suitability for pathogen testing using RT-PCR and RT-qPCR based assays (Zepeda and Verdonk, 2022).…”

“…Secondary purity assessment for extraction buffer salts and reagents contamination using the 260/230 absorption ratio indicated high amounts of reagent carryover (260/230 ratio mean= 0.93 ± 0.77, n= 6,461). Even though there are no generally accepted values for optimum 260/230 ratios for RNA extraction protocols (Cicinnati et al, 2008;Ahlfen and Schlumpberger, 2010;Gallagher, 2017;Zepeda and Verdonk, 2022), taking into consideration one of the recommended ratio values of 1.8 Imbeaud et al, 2005, 87.5% of the ratios were below that value (Figure 2B). The concentration of the isolated RNA ranged from 8.16 to 256.96 ng/mL, with a mean of 67.97 (± 33.13 ng/mL, n= 6,461), while most of the samples (78.1%) had concentrations from 25 to 100 ng/mL (Figure 2C).…”

“…The 260/230 absorption ratio can be used as a secondary measurement of RNA purity for the detection of contaminants such as buffer salts, solvents and other impurities. Even though the ratio of 1.8 has been used as a minimum RNA purity metric by some researchers (Imbeaud et al, 2005), a generally accepted range of optimum or minimum 260/230 ratio values has not been defined (Cicinnati et al, 2008;von Ahlfen and Schlumpberger, 2010;Gallagher, 2017;Zepeda and Verdonk, 2022).…”

Citrate‐Citric Acid RNA Isolation (CiAR) for Fast, Low‐Cost, and Reliable RNA Extraction from Multiple Plant Species and Tissues

“…Although the method works well with multiple tissues and plants species, different protocols are recommended for seeds (not tested yet with the CiAR method, e.g., see Oñate‐Sánchez & Vicente‐Carbajosa, 2008) or to obtain larger quantities of RNA (see Holding et al., 2007; Reyes et al., 2011; see Current Protocols article; Zepeda & Verdonk, 2021).…”