Jan 09, 2024

Public workspaceL-3 LEECH STORAGE

DOI
dx.doi.org/10.17504/protocols.io.rm7vzx234gx1/v1
  • REDI-NET Consortium1
  • 1REDI-NET Consortium
REDI-NET Consortium
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzx234gx1/v1
Protocol Citation: REDI-NET Consortium 2024. L-3 LEECH STORAGE. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx234gx1/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: November 17, 2023
Last Modified: March 15, 2024
Protocol Integer ID: 91102
Keywords: STORAGE PROCEDURE FOR UNTREATED SAMPLE, STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
Funders Acknowledgement:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: HT9425-23-C-0059
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 and HT9425-23-C-0059. 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
This protocol describes leech storage.
Guidelines
OBJECTIVE

To outline steps for properly storing field-collected leech samples and nucleic acid samples purified from these soil.

SUMMARY/SCOPE

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 leech samples and their purified nucleic acid to preserve their integrity for downstream nucleic acid extraction and/or sequencing library preparation.


MAINTENANCE OF EQUIPMENT

Decontaminate a PCR workstation by keeping the UV light on for Duration00:15:00 .

Materials
EQUIPMENT AND MATERIALS
Note
NOTE: If product number is listed, please ensure use of this or equivalent product.
ABC
Equipment / MaterialDescriptionMfg / Product #
–80°C freezerFor sample storageLocally sourced
ForcepsClean, stainlessLocally sourced
IceTo maintain cold chain during sample handlingLocally sourced
96-Well Microfuge tube racks with coverTo hold microplatesLocally sourced
KingFisher™ 96 KF microplateTo store the sampleThermoFisher, 97002540
PCR WorkstationPCR 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
Data sheetREDI-NET DCS T-3 Tick StorageREDI-NET Data Portal
Safety warnings
Attention
RISKS AND PERSONAL PROTECTION:

Gloves should be worn all the time when handling samples.
STORAGE PROCEDURE FOR UNTREATED SAMPLE
STORAGE PROCEDURE FOR UNTREATED SAMPLE

Note
NOTES:
  • Collected leeches need to be kept on cold chain all the time to prevent death and RNA degradation. Viable leeches can be temporarily stored in water at Temperature4 °C before being stored individually inTemperature-80 °C freezer. Store leeches at Temperature-80. °C until sample processing.
  • If Temperature-80 °C storage is not possible, temporarily store the leech samples in a Temperature-20 °C freezer and follow leech sample processing SOP (REDI-NET Leech Processing SOP L-2) 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 leech sample testing SOP (REDI-NET Leech Testing SOP L-4) cDNA Synthesis until finishing step 40.


Cool 96-well microfuge tube racks TemperatureOn ice .
Label 2.0 mL microfuge tubes with Sample ID.
Using clean forceps, transfer individual leech into the corresponding pre-labeled 2.0 mL microfuge tubes and put it onto the microfuge tube rack TemperatureOn ice sequentially.

Once the rack is full or all leech samples have been completed, label the rack ID with a unique ID.
Close the rack lid tightly, secure with clear Saran wrap and immediately transfer to Temperature-80 °C or Temperature-20 °C freezer.

Note
NOTES:
  • If -20ºC, total nucleic acid extraction needs to be performed preferably within 3 days.

Update the freezer inventory so samples can be tracked properly.
STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID

Note
NOTES:
  • The following procedure is to properly store total nucleic acid extracted from leech samples (including negative controls) using the 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).
  • Total nucleic acid samples need to be kept TemperatureOn ice all the time to minimize RNA degradation.
In the clean PCR workstation, carefully transfer the eluted total nucleic acid to a 96-well PCR microplate, make sure to keep samples in the exact same locations corresponding to the rack where the original ticks were stored.
Note
IMPORTANT: Mark the “A1” position of the 96-well microplate to prevent any mistakes on plate orientation.

Critical
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.
Update the freezer inventory so that samples can be tracked properly.
Protocol references