L-3 LEECH STORAGE

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.

Steps

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 4°C before being stored individually in-80°C freezer. Store leeches at -80.°C until sample processing.If -80°C storage is not possible, temporarily store the leech samples in a -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 -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 On 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 On 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 -80°C or -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

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 s 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.

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

Label the film with a unique plate ID.

10.

Immediately transfer the 96-well PCR microplate to -80°C freezer.

11.

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