Dec 17, 2024
REDI-NET FF-3 FILTH FLY STORAGE
- 1REDI-NET Consortium
- Remote Emerging Disease Intelligence - NETwork (REDI-NET)
Protocol Citation: REDI-NET Consortium 2024. REDI-NET FF-3 FILTH FLY STORAGE. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71jd1gwz/v1
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 12, 2024
Last Modified: December 17, 2024
Protocol Integer ID: 107431
Keywords: Flies, Filth flies, Storage, Total Nucleic Acid, TNA
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 filth fly 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 filth fly samples and nucleic acid samples purified from these samples.
Materials
| A | B | C | |
| Equipment / Material | Description | Mfg / Product # | |
| –80°C freezer | For sample storage | Locally sourced | |
| Ice | To maintain cold chain during sample handling | Locally sourced | |
| 96-Well Microfuge tube racks with cover | To hold the samples | Locally sourced | |
| KingFisher‱ 96 KF microplate | To store the TNA samples | ThermoFisher, 97002540 | |
| PCR Workstation | PCR cabinet with UV light | Locally sourced | |
| Clear Adhesive Film | To seal the KingFisher‱ 96 KF microplate | ThermoFisher, 4306311 | |
| Adjustable micropipettes | To handle the samples | Locally sourced | |
| Multi-channel micropipettes | 8- or 12- channel; to handle the sample | Locally sourced | |
| Nuclease-free filter tips low-retention | To ensure appropriate sample handling | Locally sourced | |
| Nuclease free microfuge tubes | 1.5 mL | Locally sourced | |
| Saran wrap | Plastic wrap; to seal rack holding sample | Locally sourced | |
| Permanent markers | To label tubes and microplates | Locally 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 00:15:00 .
STORAGE PROCEDURE FOR UNTREATED SAMPLE
STORAGE PROCEDURE FOR UNTREATED SAMPLE
Precool 96-well microfuge tube racks on ice.
Note
NOTE: Filth fly samples need to be kept on a cold chain all the time to prevent RNA degradation. The following procedure will apply only where -80ºC storage is feasible and flies are not being kept alive for research purposes.
NOTE: If -80 °C storage is not possible, temporarily store the filth fly samples in a -20 °C freezer and follow filth fly sample processing SOP (REDI-NET SOP FF-2 Filth Fly 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 -20 °C storage. To do this, follow the initial steps of the filth fly sample testing SOP (REDI-NET SOP FF-4 Filth Fly Testing) until finishing the step of “Removal of RNA”.
Using preprinted adhesive labels or permanent markers, label 1.5 mL microfuge tubes with unique sample ID.
Transfer filth sly sample into the corresponding pre-labeled 1.5 mL microfuge tubes, close the cap and put it onto the microfuge tube rack (on ice) sequentially.
Once the rack is full or all filth fly 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 -80 °C freezer.
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 filth fly 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 samples were stored. IMPORTANT: Mark the “A1” position on the 96-well microplate to avoid any mistakes on plate orientation.
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 -80 °C freezer.
Protocol references