Most of the projects we work on, whether profiling host gene expression or micobial communities, often yield at a candidate list of genes or organisms that we believe are linked to some phenotype. Increasingly, we are interested in methods and technology that let us quantitiatively measure these features, particularly in a way that is portable and could be deployed 'stall-side' in a barn just as easily as at a lab bench. For this, we've been using the Biomeme platform for end-to-end QPCR-based detection in the field
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Before starting

The protocols below detail how we’ve been using Biomeme Inc’s bulk reagents for QPCRs. Once you’ve optimized the assays using bulk reagents, assays can then be manufactured as room-temp stable QPCR assays for use in the field. For the most part, we keep our mastermix and PCR conditions the same across all assays. The variations come with the primer/probe design and optimizing extaction of DNA or RNA from different sample types. So far, we’ve had good success with the primers/probes listed below and with extraction of nucleic acid from nasal and skin swabs.

Devices

The hand-held QPCR device made by Biomeme, called the ‘three9’, accomodates 9 tubes and three colors/tube: FAM, Cy5/ATTO647N and TexasRed. ATTO647N is the brightest channel on the three9, so consider designing the target you anticipate being present in the lowest concentration and/or least sensitive PCR on that channel. FAM is the next brightest, followed by TexasRedX. If you are designing a triplex assay for use with the three9 that includes a positive control, use TexasRedX for the positive control.

Preparing mastermix

Resuspend bulk lyophilized LyoDNA, LyoRNA or LyoGreen master mix with 135 ul of PCR grade water + 18 ul of glycerol. This is a 10x stock. Addition of glyceral allows leftover mastermix to be frozen for reuse later. LyoRNA master mix contains a reverse transcriptase for QPCR assays where the target is a RNA molecule. Each resuspended vial contains enough mastermix for 60 reactions of 20ul/rxn.

Reaction set-up with LyoDNA or LyoRNA

A typical QPCR reaction on the biomeme platform contains the following components

Reagent vol (ul)
20x primer/probe 1
10x LyoDNA, LyoRNA or LyoGreen mastermix 2
isolated DNA or RNA 5-17
water qs to 20

Run set-up

  • To begin, launch the Biomeme app on the iPhone connected to your device.
  • To run any assays, you’ll need to set up a data folder by selecting the ‘data management’ button on the main screen. Name your folder, create any subfolders if necessary and save.
  • Back on the main screen, choose ‘protocol management’ to set up a protocol that will contain your thermocycling conditions and a standard curve (optional). We’ve always used the standard thermocycling conditions that come pre-set on the device as follows:
Temp (C) Time (min:sec)
45 10:00
95 10:00
95 0:15
60 1:00

Bold steps are repeated for 40 cycles total

  • Give your protocol a name and select ‘save’ in the upper right corner of the app

  • If you want to set-up a standard curve, select your protocol, scroll to the bottom, and select standard curve.

  • To begin a run, go back to the main screen of the app and choose ‘start run’. Choose your data folder, then your protocol, then select the run type (standard curve, qualitative, etc). Hit run and follow the on screen instructions to begin the run
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