RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples

Bachoon, D

doi:10.1016/s0378-1097(01)00251-8

Cited by 4 publications

(4 citation statements)

References 23 publications

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“…The longer storage time prior to RNA extraction for the September samples than the August samples during sample transportation in a freeze box and storage in a refrigerator may have resulted in higher relative eRNA concentrations from the samples in RNAlater. Our results support the technical validity in previous studies using RNAlater to preserve and reliably detect microbial, bulk, and sh eRNA in natural environments (e.g., Bachoon et al, 2001;Gorokhova, 2005;Miyata et al, 2021), and will boost the application of eRNA for the non-destructive and cost-effective monitoring of macrobial physiology at the population-and community-levels (Yates et al, 2021).…”

Section: Discussionsupporting

confidence: 87%

Simple and efficient preservation of fish environmental RNA in filtered water samples via RNAlater

Jo¹,

Matsuda

Hirohara

et al. 2022

Preprint

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Environmental RNA (eRNA) analysis has recently received attention as a better means to infer the physiological status of a community and living biotic assemblages than environmental DNA (eDNA). However, eRNA is thought to be degraded more rapidly than eDNA, increasing the risk of false-negative detection and complicating large-scale eRNA sampling in the field. In addition, the need for a deep freezer (− 80°C or below) further limits the practical application of eRNA analysis in places that are not accessible by vehicle. Here we focused on two types of reagents (RNAlater and LBP buffer) and assessed their performance for eRNA preservation. We found that very high concentrations of ayu (Plecoglossus altivelis) eRNA were quantifiable from filter samples collected from an aquarium by using RNAlater preservation at − 20°C for at least a week. Compared with the sample stored at − 20°C without any preservative, the filter samples preserved in RNAlater had eRNA concentrations that were tens to hundreds of times higher. Although further technical refinement is needed, our findings have provided valuable information to enhance the methodology for improving eRNA quality and quantity in environmental samples. This will boost the practical application of eRNA-based meta-transcriptomics targeting macro-organisms.

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Section: Discussionsupporting

confidence: 87%

Simple and efficient preservation of fish environmental RNA in filtered water samples via RNAlater

Jo¹,

Matsuda

Hirohara

et al. 2022

Preprint

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“…Ethanol might also be a suitable RNA fixative for other organisms. Bachoon et al () have suggested 96% ethanol for mRNA preservation in prokaryotes ( Pseudomonas, Synechococcus ), and 100% ethanol was successfully used to preserve viruses (influenza virus A and B, and adenoviruses) in nasal swabs at room temperature for up to 6 months (Krafft et al ). Nevertheless, cooling or freezing is advisable, especially when long‐term storage is required.…”

Section: Discussionmentioning

confidence: 99%

Ethanol: A simple and effective RNA‐preservation for freshwater insects living in remote habitats

Astrid

Egg

Füreder

2015

Limnology & Ocean Methods

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The often proposed preservation method “snap‐freezing” is rather unpractical for sampling in remote areas. Therefore, the effectiveness, feasibility and affordability of alternative fixatives were investigated. Larvae of Orthocladius sp. (Diptera, Chironomidae) were fixed in RNAlater®, RNAfix (self‐made), acetone and ethanol of two different qualities (methylated and molecular grade). Samples were first kept at room temperature for 24 h, then at −20°C for 1 week, at −80°C for 1 month and at −80°C for 6 months. Additionally, long term storage at −20°C for up to 18 months was tested for methylated ethanol and RNAfix. Subsequently evaluated RNA quality and quantity did not differ significantly between the preservation methods. Methylated ethanol, being the most economical alternative, was chosen to be tested in a field trip. Diamesa latitarsis larvae (Diptera, Chironomidae) and Baetis alpinus nymphs (Ephemeroptera, Baetidae) were collected in two remote streams at 2208 m a.s.l. Samples were cooled (∼ 4°C) for 23 up to 30 h during sampling and transportation. Organisms were identified and photographed in ethanol at room temperature and then stored at −80°C for 6 months until RNA isolation. RNA quality was high and the yield was suitable for down‐stream applications such as cDNA synthesis and real‐time quantitative PCR. Due to these results we recommend methylated ethanol as an inexpensive but reliable RNA‐fixative for sampling in remote areas even if time‐consuming species identification has to be done prior to RNA‐extraction.

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“…Tissues stored in RNAlater® for periods exceeding one month require sub-freezing conditions to maintain high-quality RNA. Many studies have confirmed the ability of RNAlater® to effectively preserve RNA in samples when following manufacturer protocols for tissue storage at frozen (-20 °C), refrigerated (4 °C), and room temperatures (21 °C) [8][9][10][11][12][13][14][15].…”

Section: Introductionmentioning

confidence: 99%

The Effects of Non-Ideal Temperature Regimes on RNA Quality from Samples Stored in RNAlater-like Buffer: An Attempt to Replicate Field Conditions

2016

J Analyt Molecul Tech

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Advances in sequencing technologies have increased the utility of RNA to many fields of biology. Ideal preservation of RNA in tissues is accomplished by flash freezing and subsequent storage at -80 °C. Buffers such as RNAlater® are commonly used to preserve RNA samples when flash freezing is not possible. RNAlater® performs optimally at subfreezing temperatures, but is also suitable for short periods at room temperature (21 °C). The effectiveness of RNAlater® when samples are stored above room temperature has not been tested. Samples collected outside of the lab are often subjected to temperatures that can exceed room temperature and fluctuate over time. Insect tissues were submerged in RNAlater-like buffer, a noncommercial RNA preservation buffer similar to commercial RNAlater, under various temperature regimes for 21 days. Tests were designed to mimic condition for tissues collected in the field and included constant temperatures of -20 °C, 0 °C, 4 °C, 21 °C, 30 °C, and temperatures that oscillated between -20 °C and 30 °C among others. RNA quality and degradation were determined by electropherograms and electrophoretic gels. Samples yielded high-quality RNA when stored in frozen (-20 °C) or cool (0 °C or 4 °C) conditions, although slightly higher RNA quality was recovered from samples stored in the 0-4 °C temperature range. RNA from samples stored at 21 °C or at oscillating temperatures of -20 °C and 30 °C were of lower quality and were unacceptable for most next-generation sequencing assays. Room temperature appears to be the critical temperature threshold above which RNA in tissues degrades rapidly. Freeze/thaw cycling was shown to mildly improve RNA quality compared to samples stored at a constant 30 °C.

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RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples

Cited by 4 publications

References 23 publications

Simple and efficient preservation of fish environmental RNA in filtered water samples via RNAlater

Simple and efficient preservation of fish environmental RNA in filtered water samples via RNAlater

Ethanol: A simple and effective RNA‐preservation for freshwater insects living in remote habitats

The Effects of Non-Ideal Temperature Regimes on RNA Quality from Samples Stored in RNAlater-like Buffer: An Attempt to Replicate Field Conditions

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