Dec 16, 2024

Version 2

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# REDI-NET L-3 LEECH STORAGE V.2

DOI

https://dx.doi.org/10.17504/protocols.io.rm7vzx234gx1/v2

REDI-NET Consortium¹

¹REDI-NET Consortium

Remote Emerging Disease...

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REDI-NET Consortium

University of Notre Dame, Naval Medical Research Center, Wal...

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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzx234gx1/v2

**Protocol Citation:** REDI-NET Consortium 2024. REDI-NET L-3 LEECH STORAGE. protocols.io
https://dx.doi.org/10.17504/protocols.io.rm7vzx234gx1/v2
Version created by **REDI-NET Consortium**
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**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:** October 22, 2024

**Last Modified:** December 16, 2024

**Protocol Integer ID:** 110563

**Keywords:** STORAGE PROCEDURE FOR UNTREATED SAMPLE, STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID, leech storage, storage this protocol, storage, protocol, redi

**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**

This protocol describes leech storage.
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# 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 00:15:00.

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# Materials

## EQUIPMENT AND MATERIALS

### Note

**NOTE:** If product number is listed, please ensure use of this or equivalent product.

<table><thead><tr><th>A</th><th>B</th><th>C</th></tr><tr><th>Equipment / Material</th><th>Description</th><th>Mfg / Product #</th></tr></thead><tbody><tr><td>-80°C freezer</td><td>For sample storage</td><td>Locally sourced</td></tr><tr><td>Forceps</td><td>Clean, stainless</td><td>Locally sourced</td></tr><tr><td>Ice</td><td>To maintain cold chain during sample handling</td><td>Locally sourced</td></tr><tr><td>96-Well Microfuge tube racks with cover</td><td>To hold microplates</td><td>Locally sourced</td></tr><tr><td>KingFisher™ 96 KF microplate</td><td>To store the sample</td><td>ThermoFisher, 97002540</td></tr><tr><td>PCR Workstation</td><td>PCR cabinet with UV light</td><td>Locally sourced</td></tr><tr><td>Clear Adhesive Film</td><td>To seal the KingFisher™ 96 KF microplate</td><td>ThermoFisher, 4306311</td></tr><tr><td>Adjustable micropipettes</td><td>To handle the samples</td><td>Locally sourced</td></tr><tr><td>Multi-channel micropipettes</td><td>8- or 12- channel; to handle the sample</td><td>Locally sourced</td></tr><tr><td>Nuclease-free filter tips low-retention</td><td>To ensure appropriate sample handling</td><td>Locally sourced</td></tr><tr><td>Nuclease free microfuge tubes</td><td>1.5 mL</td><td>Locally sourced</td></tr><tr><td>Saran wrap</td><td>Plastic wrap; to seal rack holding sample</td><td>Locally sourced</td></tr><tr><td>Permanent markers</td><td>To label tubes and microplates</td><td>Locally sourced</td></tr><tr><td>Data sheet</td><td>REDI-NET DCS T-3 Tick Storage</td><td>REDI-NET Data Portal</td></tr></tbody></table>
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# Troubleshooting

## Safety warnings

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**RISKS AND PERSONAL PROTECTION:**

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

1

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 ⌀ 4 °C before being stored individually in ⌀ -80 °C freezer. Store leeches at ⌀ -80. °Cuntil sample processing.

* If ⌀ -80 °Cstorage is not possible, temporarily store the leech samples in a ⌀ -20 °Cfreezer 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 ⌀ -20 °Cstorage. 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 ⌀ On ice.

2

Label ⌀ 2.0 mL microfuge tubes with Sample ID.

3

Using clean forceps, transfer individual leech into the corresponding pre-labeled ⌀ 2.0 mL microfuge tubes and put it onto the microfuge tube rack ⌀ On ice sequentially.

4

Once the rack is full or all leech samples have been completed, label the rack ID with a unique ID.

5

Close the rack lid tightly, secure with clear Saran wrap and immediately transfer to ⌀ -80 °C or ⌀ -20 °C freezer.
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Note

NOTES:

* If °C -20, total nucleic acid extraction needs to be performed preferably within 3 days.

6. Update the freezer inventory so samples can be tracked properly.

STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID

7

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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 On 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.

8. Cover the 96-well PCR microplate with adhesive film to prevent spill over or contamination.

9. Label the film with a unique plate ID.

10. Immediately transfer the 96-well PCR microplate to -80 °C freezer.
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11 Update the freezer inventory so that samples can be tracked properly.

Protocol references

REFERENCES

REDI-NET Overview Summary