Research Questions

Background

Transcriptomes are more cost-effective than genomes and can serve the same purposes in many cases (Yang et al. 2017). However, there are many difficulties involved in obtaining and preserving tissue for RNA-seq. Most of these come from the need to immediately flash freeze tissue in liquid nitrogen and store it at low temperatures. This makes remote field work more arduous and expensive, and excludes many areas where liquid nitrogen is not available.

Technological advances have extended the time that samples can stay at room temperature, but many protocols still require freezer storage. It is yet unclear how RNA degrades in tissue that is not preserved using common methods. Multiple studies (most recently He et al. 2022 and Ruiz-Vargas et al. 2023) have shown the viability of silica-dried plant tissue for RNA extraction. Some studies have detected RNA survival under typical herbarium preservation methods and the survival of seed RNA for centuries (see Selected Studies table below).

The observed transcriptomic sampling gap shows geographic bias, potentially due to the expenses involved in flash-freezing and long-term cold storage. The use of dry, room temperature plant samples for transcriptomics may greatly expand the number of plant species that can be sequenced. We may be able to extend our transcriptomic sampling to the wealth of plant samples preserved in herbaria. The concept of herbariomics has thus far only encompassed DNA studies, but RNA from many herbarium samples may still be viable for many uses.

Further, the global climate cost of long-term cold storage should not be understated. Preserving, transporting, and storing plant samples at −80°C is costly for universities and for the environment. Each −80°C freezer consumes about 20 kWh/day, approximately as much energy as an average U.S. home (U.S. Federal Energy Management Program). Even small temperature adjustments from −80°C to −70°C for less sensitive samples can reduce cost and carbon footprint. Given this, it is important to understand the impact preservation methods have on the utility of plant samples.

A thorough investigation of the viability of plant RNA from dried samples has not been performed, although the ability to sample such specimens could greatly increase the number of plants that could be studied using transcriptomics. Silica drying can lower the burden of preservation and may still allow the production of usable transcriptome assemblies (Ruiz-Vargas et al. 2023; He et al. 2022). In the future, we hope to examine the utility of multiple preservation methods for transcriptomics and the effect of the preservation method on gene recovery in an experimental fashion.

Figure 1a: Total global vascular plant species richness

1a) Global species occurrence data were collected from the RBIEN database. Each species in the Kew World Checklist of Vascular Plants (WCVP) was searched in RBIEN (WCVP n=356,618, within RBIEN n= 174,106). Lat/long pairs (n=22,881,401) were downloaded and rasterized over a 4x4 latitude by longitude grid.

Figure 1b: Richness for vascular plant species with a sequenced transcriptome

1b) The records for transcriptome sequenced species which occurred in the SRA (n= 4,653 valid species were present in RBIEN, out of 6,966) were collected. This returned 6,441,980 lat/long pairs.

Figure 1c: Proportion of species with a sequenced transcriptome

1c) An approximate measure of completeness was calculated by taking the number of species with transcriptomes sequenced, compared to the predicted number of species in a grid cell.

Figure 2: RNA-seq Species Diversity Over Time

2a) The increase in RNA-seq species diversity within vascular plants over time. The NCBI SRA reports statistics from uploaded RNA-seq runs, which were filtered by name to species level and referenced to currently accepted scientific names using the rgbif taxonomic backbone.

Year 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022
New species 8 33 76 143 187 224 366 716 715 1,747 1,164 1,186 1,456 1,891 2,219

2b) The cumulative summation of unique vascular plant species that have been sequenced by year. Each species is only added to the total on the year it is first sequenced. A total of 6,966 unique and valid species within vascular plants have been sampled and uploaded to the NCBI SRA from 2008 to 2022, representing 406,177 records.

Year 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022
Cumulative species 8 38 102 213 335 466 692 1,099 1,511 2,745 3,378 3,960 4,654 5,753 6,966

Figure 3: RNA from Silica-Dried Tissue

RNA can be obtained from six-month old silica-dried tissue.

3a) Pitcairnia jimenezii (family Bromeliaceae) — one of the species used in the study by Ruiz-Vargas et al. 2023.

3b) From Ruiz-Vargas et al. 2023 (bioRxiv). Leaf tissue from 19 samples from the genus Pitcairnia (family Bromeliaceae) was stored in silica gel for three to six months at room temperature, then RNA was extracted and sequenced. There is no strong trend associating total RNA (ng) or RIN and final predicted gene count.

Selected Studies Inspiring This Research

Study Organism Type Preservation Sample age / origin RNA detection method
Ruiz-Vargas et al. 2023 Pitcairnia spp. Plant Silica gel at RT 3–6 months old RNA sequencing; assembly; RIN; total RNA
He et al. 2022 Multiple Plant Silica dried then frozen 0–2 years old RNA sequencing; assembly; total RNA concentration; RIN; OD 260/280; OD 260/230
Hamim 2022 Multiple plant virus Plant RNAlater at changing temperatures 2–2.5 months old cDNA synthesis; RNA sequencing; assembly; PCR; gel electrophoresis; spectrophotometry
Mark et al. 2022 Multiple plant virus Plant Herbarium dry at RT Up to 56 months old Gel DNA/RNA separation; cDNA synthesis; PCR
Rieux et al. 2021 African cassava mosaic virus Plant virus Herbarium dry at RT Originally from 1928 sRNA sequencing
Jimenez et al. 2021 Multiple Plant virus Silica gel at RT or cold storage 1.5–9 months RNA concentration; OD260nm/OD280nm; total nucleic acid yield
De Wever et al. 2020 Theobroma cacao L. Plant Seed at multiple temperatures 0–5 weeks old Qubit RNA HS; fragment analyzer; RQN; RT qPCR
Fleming et al. 2019 Multiple; >40 species Plant Seed at multiple temperatures Originally from 1959–2017 Germination assay; RIN; RNA concentration
Fleming et al. 2017 Glycine max cv. Williams 82 Plant Seed Originally from 1989–2015 Germination assay; RIN; fragment size analysis; RNA yield per seed
Mangeot-Peter et al. 2016 Cannabis sativa Plant Ethanol 1–8 days old RIN; PCR; Bioanalyzer
Hartung et al. 2015 Citrus leprosis virus Plant virus Herbarium dry at RT Originally from 1932–1967 sRNA sequencing; assembly; Qubit; Bioanalyzer
García-Baldenegro et al. 2015 Vitis vinifera cv. Flame Seedless Plant Freezing then lyophilization 6 months at −80°C then 0–6 weeks lyophilized RT-PCR; A260/A280; electrophoresis; Bioanalyzer; cDNA synthesis
Smith et al. 2014 Barley Stripe Mosaic Virus Plant virus Seed 1264 ± 150 CE sRNA sequencing; assembly
Fordyce et al. 2013 Zea mays spp. mays Plant Seed 1290 ± 23 CE PCR; long and short read sequencing of cDNA; assembly
Sallon et al. 2008 Phoenix dactylifera Plant Seed 295 ± 47 CE RAPD
Natarajan et al. 2000 Solanum tuberosum Plant FTA® Card 5 days old RT-PCR
Rollo 1985 Lepidium sativum L. Plant Seed 1400 BCE Spot hybridization

Future Directions

A thorough investigation of the viability of plant RNA from dried samples has not been performed, although the ability to sample such specimens could greatly increase the number of plants that could be studied using transcriptomics. Silica drying can lower the burden of preservation and may still allow the production of usable transcriptome assemblies (Fig. 3a; Ruiz-Vargas et al. 2023; He et al. 2022). In the future, we hope to examine the utility of multiple preservation methods for transcriptomics and the effect of the preservation method on gene recovery in an experimental fashion.

Acknowledgements

This work was supported by startup funds from the University of Illinois at Chicago to J.W., as well as National Science Foundation GRFP award 2236870 to A.T. and IOS 2109716 to D.L.

Contact

Alexa Tyszka  ·  atyszka2@uic.edu  ·  atyszka.org