RNA preservation & RNA extraction protocol suitable for field collection of coral samples

1 RNA preservation & RNA extraction protocol suitable for field collection of coral samples Christian R Voolstra*, Gabriela Perna, Rachel Alderdice Department of Biology, University of Konstanz, Konstanz, Germany *Corresponding author, [email protected] Summary The ability to collect samples suitable for RNA extraction in remote field settings is typically hampered by the need for immediate processing or freezing of samples in liquid nitrogen for preservation of nucleic acid integrity. To this end, a range of RNA preservation buffers are available, compatible with ambient temperature transport and subsequent RNA extraction. Here we tested two commonly available RNA storage buffers, RNAlater from Thermo Fisher Scientific and DNA/RNA Shield from Zymo Research, with subsequent RNA extraction using the RNeasy Mini Kit from Qiagen. We assessed three common coral genera (Acropora, Pocillopora, Porites) reported to yield different levels of RNA quality and quantity, partially due to varying levels of mucus that co-purifies with RNA. All samples were collected in the field and preserved at site (on the boat) with storage at ambient temperature, followed by +4°C and -20°C storage, before subsequent RNA extraction. We found DNA/RNA Shield in combination with the RNeasy Mini Kit to yield non-degraded high quality total RNA. The lytic activity of the DNA/RNA Shield buffer facilitates the separation of the coral tissue from the skeleton. At the same time, minimizing the amount of storage buffer is important to obtain sufficiently concentrated RNA for direct use in downstream applications (e.g., RNA-Seq). In summary, we developed a protocol for coral RNA preservation and RNA extraction suitable for application in remote field settings that yield sufficiently concentrated, high quality RNA for direct use in downstream molecular applications. Materials & Methods Sample collection In December 2021, fragments from adult colonies of Porites sp. Acropora sp., and Pocillopora sp. were collected from Al Fahal reef (22.304909, 38.965029) in the central Red Sea, Saudi Arabia. From each species, fragments for RNAlater and DNA/RNA Shield from 3 replicate colonies were sampled using a hammer and chisel on SCUBA at depths between 3-8 m. Upon return to the boat, samples were placed in 25 ml tubes and either immersed in RNAlater (Thermo Fisher Scientific) or DNA/RNA Shield (Zymo Research). All corals were collected under permits from the Saudi Coastguard Authority, issued under the auspices of the King Abdullah University of Science and Technology (KAUST). Sample storage Upon return to shore, samples were cut into smaller pieces and transferred to 5 ml tubes for longterm storage. The original used storage buffer was re-added to the samples and, after incubation at room temperature for ~2 hours, samples were stored at 4ºC for transport to the University of Konstanz, Germany where they were stored at -20ºC until processing. 2 RNA isolation RNA isolation was performed using the Qiagen RNeasy Mini Kit in combination with the Qiagen QIAcube Connect automated extraction robot. Coral samples were thawed at room temperature and the coral tissue sprayed off using airflow from a sterile 1000 µl pipette tip connected via a rubber hose to a benchtop air pressure valve. Coral samples stored in RNAlater were removed from the storage tubes with sterile forceps and tapped gently to remove any excess solution. Following this, they were placed into individual sterile whirl-pak bags (Whirl-Pak) and 1000 µl of the RNeasy Mini Kit RLT buffer added to the fragment. The tissue was then sprayed off using the airflow for a maximum of 3 minutes and collected into the bottom of the whirl-pak bag. The resulting tissue slurry was transferred into a round bottom 2 ml tube and homogenized using a homogenizer (Polytron PT 1200 E, Kinematica) before being centrifuged at full speed for 3 mins. Following this, 300 µL of the supernatant were transferred into a new round bottom 2 mL tube, and RNA was extracted using the RNeasy Mini Kit with a DNase digestion step following the manufacturer’s instructions. Samples were eluted in 50 µl RNase-free water. Coral samples stored in DNA/RNA Shield were transferred along with the full volume of the buffer solution into sterile whirl-pak bags, and the fragments were sprayed until all tissue was removed. As evidenced by coloration of the buffer over storage time, part of the coral tissue was already lysed in the storage buffer, due to its lytic activity (Figure S1). The resultant slurry was transferred back into the original 5 ml storage tube and homogenized using a homogenizer (Polytron PT 1200 E, Kinematica). Following this, 300 µl of homogenized coral tissue were transferred to a 1.5 mL tube and 300 µl of RNeasy Mini Kit RLT buffer were added with subsequent vortexing, before centrifugation at full speed for 3 mins. The supernatant was transferred into a round bottom 2 mL tube. RNA was extracted using the RNeasy Mini Kit with a DNase digestion step following the manufacturer’s instructions. Samples were eluted in 30 µl RNase-free water. RNA was quantified using the QIAxcel RNA QC Kit (Qiagen) on the QIAxcel Advanced System (Qiagen). RNA quality was assessed using the Agilent 4150 TapeStation with the RNA ScreenTape assay (Agilent Technologies). RNA precipitation Given that the amount of DNA/RNA Shield buffer determines the final RNA concentration of the extraction (as the entirety of the buffer needs to be used due to its inherent lytic activity), we concentrated all RNA extractions using 2.5 volumes of 100% EtOH, 0.1 volumes of 3M sodium acetate pH 5.2, and 1 μL of glycogen (20 mg/mL) to increase yield (Walker & Lorsch, 2013). This was done to stay within recommended RNA concentrations for the RNA ScreenTape assay (≥ 25 - 500 ng total RNA in 1 µL). Samples were incubated for at least 2 hours at -80°C. Precipitated RNA was pelleted by centrifugation at 12,000 g for 15 mins at 4°C. The supernatant was removed, the RNA pellet washed with 2.5 volumes of 70% ethanol, air dried for 10 mins at room temperature, and resuspended in 10 µL of RNase-free water for RNAlater samples and in 6 µL for DNA/RNA Shield samples. 3 Results Three species of corals (Table 1) were preserved using two treatments (n = 3 per species per treatment): RNAlater and DNA/RNA Shield. All samples were then processed using Qiagen’s RNeasy Mini Kit. With regard to RNA quantity, total RNA was obtained from all coral samples, although the relative amounts of extracted total RNA exhibited a genus-/species-specific effect with Pocillopora sp. samples yielding highest RNA amounts, followed by Acropora sp., followed by Porites sp., irrespective of the storage buffer used (Table 1). With regard to RNA quality, samples preserved in DNA/RNA Shield yielded consistently nondegraded RNA, as denoted by the sharp 18S and 28S RNA peaks and also consistently higher RIN values, in comparison to samples preserved in RNAlater (Figure 1). Discussion The genomic revolution has made the molecular analysis of large cohorts of field samples feasible, both in terms of processing and cost (Ungerer, Johnson, & Herman, 2008). This necessitates the need for sample preservation in remote field settings, either for DNAor RNAbased applications (Gray, Pratte, & Kellogg, 2013; Voolstra, Quigley, et al., 2021). In particular, RNA is very unstable at room temperature and degrades in less than an hour. A range of RNA preservation buffers are now available that are compatible with ambient temperature storage and transport. Here we assessed RNAlater (Thermo Fisher Scientific) and DNA/RNA Shield (Zymo Research), two commonly available RNA storage buffers, for their suitability for coral RNA preservation using the RNeasy Mini Kit (Qiagen). For the three tested species (Pocillopora sp., Porites sp., Acropora sp.), DNA/RNA Shield yielded largely undegraded, intact total RNA, as evidenced by the presence of clear and distinct 18S and 28S rRNA bands. Also, RIN numbers were consistently higher for DNA/RNA Shield preserved specimens. Of note, the lytic activity of the DNA/RNA Shield buffer necessitates that the entire storage buffer volume and the preserved specimen need to be considered for RNA extraction, as already lysed cells in the buffer solution will otherwise be unaccounted. Therefore, it is important to minimize storage buffer volume in order to achieve the total RNA concentrations required for subsequent molecular applications. For our samples, Pocillopora and Acropora yielded sufficiently highly concentrated total RNA for direct downstream application. However, Porites sp. is known to harbor high amounts of mucus that interferes with nucleic acid isolations and typically results in low yields (Bouchard, Michaels, & Brown-Harding, 2020; Robbins et al., 2019). This was confirmed for our samples and irrespective of the preservation buffer used (Table 1). To increase total RNA concentrations, samples were hence precipitated to achieve concentrations recommended for the TapeStation System. As evident from Table 1 and Figure 1, this only worked for some of the Porites sp. samples (in the other cases the precipitated pellet might have been lost resulting in loss of RNA). As such, this added complication should be 4 avoided if possible. We have found that storing samples in plastic bags (e.g., Whirl Paks) allows the use of very low volumes of storage buffer (~2-3 mL) to achieve higher total RNA concentrations. Plastic bags also accommodate the fact that coral fragments typically come in shapes and sizes that makes them challenging to fit in a 25mlor 50ml-conical tube opening (Figure S2). Of additional note, spraying off tissue from the underlying coral skeleton is typically not an easy endeavor and requires significant amounts of time and should typically be conducted under a hooded area or while wearing a face mask and safety goggles, due to the allergic properties of coral tissue (Miguel-Gomez & Fonda-Pascual, 2016). We have found that spraying off tissue from fragments immersed in DNA/RNA Shield works much better in that the tissue and polyps typically detach very easily from the underlying skeleton (Figure S3), which saves processing and minimizes exposure time. At large, the development of standardized protocols and procedures facilitates the comparison of experimental and field data (meta-analyses) (Grottoli et al., 2021; Voolstra, Suggett, et al., 2021), in particular for gene expression-based comparisons that were shown to be affected by sample collection procedure and RNA isolation method (Asare et al., 2008; Scholes & Lewis, 2020). Prompted by the large meta-analysis of expression data as part of the Paul G. Allen Family Foundation Project “Search for corals with natural resilience to climate change” (aka “Global Search”) (link, link) using the Coral Bleaching Automated Stress System (CBASS) (Voolstra et al., 2020) with subsequent molecular analyses (Voolstra, Valenzuela, et al., 2021), we here developed a protocol for RNA isolation that is compatible with collection of samples in remote field settings that we hope can be applied and will be used widely. 5 Table 1. Overview of samples and RNA extraction methods and yields. Abbreviations: Poc = Pocillopora sp., Acr = Acropora sp., Por = Porites sp.; Rep. = Replicate; RIN = RNA Integrity Number Species Rep. Storage Buffer Input/amount used for RNA extraction Elution volume [µl] Conc. [ng/µl] Conc. after precipitation [ng/µl] Total RNA amount [ng] RIN Poc 1 RNAlater Sprayed off tissue in 1000 µl RLT; used 350 µl for RNA extraction 50 169.84 2112.57 21,125.70 5.0 Poc 2 50 271.04 2592.95 25,929.50 4.9 Poc 3 50 278.25 2492.19 24,921.90 - Por 1 RNAlater Sprayed off tissue in 1000 µl RLT; used 350 µl for RNA extraction 50 2.85 192.89 1,928.90 2.8 Por 2 50 2.97 45.00 450.00 2.5 Por 3 50 0.32 9.45 94.50 - Acr 1 RNAlater Sprayed off tissue in 1000 µl RLT; used 350 µl for RNA extraction 50 76.22 1151.87 11,518.70 3.5 Acr 2 50 107.20 1252.85 12,528.50 5.1 Acr 3 N/A N/A N/A N/A N/A Poc 1 DNA/RNA Shield Sprayed off tissue using initial volume of 5ml DNA/RNA Shield; used 300 ul of DNA/RNA Shield + 300 ul RLT buffer for RNA extraction 30 153.84 467.48 2,804.88 8.6 Poc 2 30 266.94 706.44 4,238.64 7.1 Poc 3 30 535.50 406.00 2,436.00 6.6 Por 1 DNA/RNA Shield Sprayed off tissue using initial volume of 5ml DNA/RNA Shield; used 300 ul of DNA/RNA Shield + 300 ul RLT buffer for RNA extraction 30 7.27 3.00 18.00 - Por 2 30 18.21 44.88 269.28 7.5 Por 3 30 1.34 4.53 27.18 - Acr 1 DNA/RNA Shield Sprayed off tissue using initial volume of 5ml DNA/RNA Shield; used 300 ul of DNA/RNA Shield + 300 ul RLT buffer for RNA extraction 30 88.06 23.10 138.60 5.8 Acr 2 30 53.12 61.58 369.48 7.1 Acr 3 30 18.12 85.84 515.04 5.3 6 Figure 1. Total RNA profiles of selected samples (refer to Table 1 for sample details) run on an Agilent TapeStation. In all cases DNA/RNA Shield samples exhibit distinct 18S and 28S rRNA bands, indicative of no/minor RNA degradation. For 3 of the Porites sp. samples (Por3 RNAlater, Por1 DNA/RNA Shield, Por3 DNA/RNA Shield), the concentrations were below recommended concentrations (at least 25ng/µL). 7 Supplementary Figures Figure S1. Porites sp. fragment in RNAlater (left) and DNA/RNA Shield (right), illustrating the lytic activity of the DNA/RNA Shield buffer as illustrated by the brownish color of the buffer. Figure S2. Coral samples immersed in DNA/RNA Shield buffer in a zip-tied Whirl-Pak bag. The buffer shows coloration indicating the ongoing tissue lysis, which considerably facilitates spraying off the tissue from the skeleton. 8 Figure S3. Coral tissue and polyps typically detach easily from the underlying skeleton when spraying off fragments that were stored in DNA/RNA Shield buffer. Shown is the coral fragment on the left, the 1000 µl pipette tip connected to a benchtop air pressure valve on the top right, and the sprayed off polyps and tissue in DNA/RNA Shield buffer in the bottom right of the Whirl-Pak bag. The detached, sprayed-off polys look similar to chia seeds. 9 Protocol (step-by-step): DNA/RNA Shield buffer-based sample collection, storage, and processing for RNA isolation of coral field samples Resources/Read before you start - DNA/RNA Shield - RNeasy Mini Handbook Coral sample collection 1. Collect branching coral species (e.g., Acropora sp.) with hammer and chisel or clippers, collect massive coral species (e.g., Porites sp.) with underwater drill and core drill bit (e.g., link) 2. Place specimens into collection bags, make sure the bags are filled with seawater. 3. Upon return to the boat/shore place the sample bags inside a cooler box filled with seawater until further processing. DNA/RNA Shield buffer-based coral sample storage 1. Transfer coral samples to whirl-pak bags (e.g., link) and add 2-3 mL of DNA/RNA Shield buffer (use the minimum amount of buffer to cover the sample in order to maximize RNA concentration). 2. Use a (reusable) cable tie to seal the whirl-pak bag so that the coral fragment is secured and fully submerged in the DNA/RNA Shield buffer (Figure S2). 3. Store at RT/4ºC/-20°C until further processing (colder temperatures increase storage time: 35 - 40°C ≤ 7 days; 4°C - 25°C ≥ 30 days; ≤ -20°C = indefinite) Coral sample processing 1. Spray off coral tissue into the storage whirl-pak bag filled with DNA/RNA Shield using airflow from a sterile 1000 µL pipette filter-tip connected via a rubber hose to a bench top air pressure valve or an airbrush compressor set, until all of the coral tissue is removed from the skeleton (Figure S3). 2. Transfer tissue slurry with accompanying DNA/RNA Shield buffer into a screw-cap cryogenic viable and store at RT/4ºC/-20°C until further processing. Coral RNA isolation using the RNeasy Mini Kit 1. If applicable: defrost samples at room temperature. 2. Homogenize the tissue slurry with a homogenizer (e.g., Polytron PT 1200 E with a 3 mm dispersing aggregate, e.g. link) NOTE: incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy Mini kit spin column. Recommended minimum speed using disperser above is ~70% of max. NOTE: We use the RNeasy Mini Kit (link) and follow procedures based on the “Protocol: Purification of Total RNA from Animal Tissues” and the “Optional On-Column DNase Digestion with the RNAse-Free DNase Set” (Appendix D) NOTE: Read ‘Important points before starting’ in the RNeasy Mini Handbook.