Dec 16, 2024

Public workspaceREDI-NET F-3 FECES STORAGE V.2

DOI
dx.doi.org/10.17504/protocols.io.261ge572dg47/v2
  • 1REDI-NET Consortium
  • Remote Emerging Disease Intelligence - NETwork (REDI-NET)
REDI-NET Consortium
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DOI: dx.doi.org/10.17504/protocols.io.261ge572dg47/v2
Protocol Citation: REDI-NET Consortium 2024. REDI-NET F-3 FECES STORAGE. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge572dg47/v2Version created by REDI-NET Consortium
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 11, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 115704
Keywords: Feces, Total Nucleic Acid, TNA, Storage
Funders Acknowledgements:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: HT9425-23-C-0059
USAMRAA
Grant ID: HT9425-24-C-0072
Disclaimer
This work is supported by the US Army Medical Research and Development Command under Contract No.W81XWH-21-C-0001, W81XWH-22-C-0093, HT9425-23-C-0059 and HT9425-24-C-0072. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army or Navy position, policy or decision unless so designated by other documentation.
Abstract
The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on storage of fecal samples and their purified nucleic acid to preserve their integrity for downstream nucleic acid extraction and/or sequencing library preparation.
Guidelines
To outline steps for properly storing feces samples and nucleic acid samples purified from these samples.
Materials

ABC
Equipment / MaterialDescriptionMfg / Product #
–80°C freezer For sample storageLocally sourced
IceTo maintain cold chain during sample handlingLocally sourced
96-Well Microfuge tube racks with coverTo hold the samplesLocally sourced
KingFisher‱ 96 KF microplateTo store the TNA samplesThermoFisher, 97002540
PCR Workstation PCR cabinet with UV lightLocally sourced
Clear Adhesive FilmTo seal the KingFisher‱ 96 KF microplateThermoFisher, 4306311
Adjustable micropipettesTo handle the samplesLocally sourced
Multi-channel micropipettes8- or 12- channel; to handle the sampleLocally sourced
Nuclease-free filter tips low-retentionTo ensure appropriate sample handlingLocally sourced
Nuclease free microfuge tubes1.5 mLLocally sourced
Saran wrapPlastic wrap; to seal rack holding sampleLocally sourced
Permanent markersTo label tubes and microplatesLocally sourced

Safety warnings
RISKS AND PERSONAL PROTECTION:

Gloves should be worn all the time when handling samples.
Before start
MAINTENANCE OF EQUIPMENT:
Decontaminate a PCR workstation by keeping the UV light on for Duration of Duration00:15:00 .

STORAGE PROCEDURE FOR UNTREATED SAMPLE
STORAGE PROCEDURE FOR UNTREATED SAMPLE
Precool 96-well microfuge tube racks on ice.

Note
NOTE: Fecal samples need to be kept on cold chain all the time to prevent RNA degradation. The following procedure will apply only where Temperature-80 °C storage is feasible.

NOTE: If Temperature-80 °C storage is not possible, temporarily store the fecal samples in a Temperature-20 °C freezer and follow fecal sample processing SOP (REDI-NET SOP F-2 Feces Processing) as soon as possible for total nucleic acid extraction. Subsequently, use a portion of the total nucleic acid and reverse- transcribe RNA into cDNA for Temperature-20 °C storage. To do this, follow the initial steps of the fecal sample testing SOP (REDI-NET SOP F-4 Feces Testing) until finishing the step of “Removal of RNA”.


Temperature
Using preprinted adhesive labels or permanent markers, label Amount1.5 mL microfuge tubes with unique sample ID.

Transfer fecal sample into the corresponding pre-labeled Amount1.5 mL microfuge tubes and put it onto the microfuge tube rack (on ice) sequentially.

Pipetting
Once the rack is full or all fecal samples have been completed, label the rack with a unique rack ID.
Close the rack lid tightly, secure with clear saran wrap and immediately transfer to Temperature-80 °C freezer.

Enter the storage location in the REDI-NET Data Portal using the Sample Storage tab. See Guidance on REDI-NET Sample Storage Time QRG.
STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID

Note
NOTE: The following procedure is to properly store total nucleic acid extracted from fecal samples (including negative controls) using KingFisher nucleic acid purification system. The eluted total nucleic acid will be in either 96-well microplate (Flex model) or elution strip (Duo Prime model).

NOTE: Total nucleic acid samples need to be kept on ice all the time and as short as possible to minimize RNA degradation.
In the clean PCR workstation, carefully transfer the eluted total nucleic acid to a 96-well PCR microplate, making sure to keep samples in the exact same locations (e.g., A1 to A1, A2 to A2…) corresponding to the rack where the original fecal samples were stored.

IMPORTANT: Mark the “A1” position on the 96-well microplate to avoid any mistakes on plate orientation.
Pipetting
Cover the 96-well PCR microplate with adhesive film to prevent spill over or contamination.
Label the film with a unique plate ID.
Immediately transfer the 96-well PCR microplate to Temperature-80 °C freezer.

Temperature
Protocol references