Oct 28, 2024

Public workspaceA Portable Device for Lab-Free, Versatile Nucleic Acid Extraction - Protocol

DOI
dx.doi.org/10.17504/protocols.io.kxygxyj4wl8j/v1
  • Anthony J Politza1,
  • Tianyi Liu1,
  • Aneesh Kshirsagar1,
  • Ming Dong1,
  • Md. Ahasan Ahamed1,
  • Weihua Guan1
  • 1The Pennsylvania State University
Anthony J Politza
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DOI: dx.doi.org/10.17504/protocols.io.kxygxyj4wl8j/v1
Protocol Citation: Anthony J Politza, Tianyi Liu, Aneesh Kshirsagar, Ming Dong, Md. Ahasan Ahamed, Weihua Guan 2024. A Portable Device for Lab-Free, Versatile Nucleic Acid Extraction - Protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxyj4wl8j/v1
Manuscript citation:
Politza, AJ et. al. Development and Validation of a Portable Device for Lab-Free Versatile Nucleic Acid Extraction. Biotechniques, 2024.
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
As of Oct. 2024, this protocol has been tested and validated for use with viral RNA from plasma or saliva, as well as for miRNA from saliva.
Created: October 14, 2024
Last Modified: October 28, 2024
Protocol Integer ID: 109832
Keywords: Sample preparation, Nucleic Acid Testing, Portable Sample Prep, Battery-powered, Field-deployable, Versatile RNA Extraction
Funders Acknowledgement:
National Science Foundation
Grant ID: 2319913
National Science Foundation
Grant ID: 2045169
National Institutes of Health
Grant ID: R33AI147419
National Institutes of Health
Grant ID: R33HD105610
USDA
Grant ID: NIFA 2022-11225
Abstract
To enable faster, easier extraction of viral RNA outside of traditional laboratories, we use a custom, automated, and portable centrifuge for processing Qiagen QIAamp RNA extractions.

Viral RNA is eluted into approx. 60 µl of nuclease free water that can then be analyzed by downstream testing (PCR, gel electro, Qubit). Our results showed very strong agreement with benchtop extractions and probit analysis demonstrated a limiting extraction concentration of 0.75 cp/µl at 95% confidence.
Guidelines
This has now been tested on a variety of patient samples including HIV in plasma, SARS-CoV-2 in saliva, SARS-CoV-2 in VTM (from nasopharygeal swab), and miRNA in saliva (as of October 15,2024).
Materials
RNA EXTRACTION
  • Qiagen - QIAamp Viral RNA Mini Kit

PRIMERS
  • Primers were purchased from IDT.

RT-PCR
  • Applied Biosystems - Fast Virus 1-Step Master Mix
Protocol materials
ReagentQIAamp Viral RNA Mini KitQiagenCatalog #52904
Step 2
ReagentApplied Biosystems™ TaqMan™ Fast Virus 1-Step Master Mix for qPCR Applied Biosystems (ThermoFisher Scientific)Catalog #44-444-32
Step 39
Safety warnings
Please follow standard health and safety guidelines when working with viral samples.
Sample Collection
Sample Collection
Label a Amount1.5 mL tube and add each Sample50 µl Sample .


Note
The following reagent volumes and steps are assuming a 50 µl starting sample.



Note
If you need to process more than 50 µl, increase the lysis buffer and ethanol proportionally to your new sample volume (550 µl : 50 µl).
A maximum of 750 µl will fit into the top of the mini-column, therefore if you increase sample volume you will need to add additional repetitions of the RNA/DNA binding steps.
**Please see the manufacturer's manual for additional details.**

Gather ReagentQIAamp Viral RNA Mini KitQiagenCatalog #52904

RNA/DNA Lysis
RNA/DNA Lysis
Add Amount550 µL Lysis buffer to the Sample50 µl Sample
Mix well by vortexing or pipetting.
Incubate for Duration00:10:00 atTemperatureRoom temperature

Expected result
Amount600 µL DNA Lysate


10m
Power On Portable Centrifuge
Power On Portable Centrifuge
Turn on the power switch located on the rear of the device.

Expected result
A bright red LED should illuminate on the front of the device, indicating the device is ready to be used.

Note
Our device is pre-programmed to manage the spin down steps and automatically adjust the timings depending on the appropriate step. Example: Step 1. 60s Step 2. 60s etc.

**At this time the use of other devices is not recommended or tested with this protocol. We imagine mini-centrifuges operating on external battery power could work as an alternative if they maintain higher than 1750g while spinning. (Can check with equation listed below)**




RNA/DNA Binding
RNA/DNA Binding
Add Amount550 µL Ethanol (95%) to the Amount600 µL DNA Lysate

Expected result
Amount1150 µL DNA Lysate


Mix well and pipette Amount750 µL DNA Lysate / 1150µl into the top of the QIAamp Mini Spin Column.

Place the entire QIAamp Mini Spin Column & tube into the portable centrifuge.
Close the lid of the device, check the red LED is illuminated, and push the front button beside the LED.

Note
If the red LED is not illuminated check that Step 4 was completed, and that the device was charged overnight.


Wait Duration00:01:00 for the liquid to spin down.

1m
Remove the QIAamp Mini Spin Column & discard the waste tube.
Insert the QIAamp Mini Spin Column into a fresh collection tube.
Repeat Steps 7 -> 12.
RNA/DNA Wash 1
RNA/DNA Wash 1
Add Amount500 µL Buffer AW1 to the top of QIAamp Mini Spin Column.

Place the entire QIAamp Mini Spin Column & tube into the portable centrifuge.
Close the lid of the device, check the red LED is illuminated, and push the front button beside the LED.
Wait Duration00:01:00 for the liquid to spin down.

Remove the QIAamp Mini Spin Column & discard the waste tube.
Insert the QIAamp Mini Spin Column into a fresh collection tube.
RNA/DNA Wash 2
RNA/DNA Wash 2
Add Amount500 µL Buffer AW2 to the top of QIAamp Mini Spin Column.

Place the entire QIAamp Mini Spin Column & tube into the portable centrifuge.
Close the lid of the device, check the red LED is illuminated, and push the front button beside the LED.
Wait Duration00:01:00 for the liquid to spin down.

Remove the QIAamp Mini Spin Column & discard the waste tube.
Insert the QIAamp Mini Spin Column into a fresh collection tube.
RNA/DNA Drying
RNA/DNA Drying
Add Amount500 µL Ethanol (95%) to the top of QIAamp Mini Spin Column.

Place the entire QIAamp Mini Spin Column & tube into the portable centrifuge.
Close the lid of the device, check the red LED is illuminated, and push the front button beside the LED.
Wait Duration00:03:00 for the liquid to spin down.

3m
Remove the QIAamp Mini Spin Column & discard the waste tube.
Insert the Mini Column into a fresh Amount1.5 mL tube and label accordingly.

RNA/DNA Elution
RNA/DNA Elution
Add Amount80 µL Nuclease Free Water to the top of QIAamp Mini Spin Column. Be sure to drop liquid into the center of the column membrane.
Note
In this protocol we used Nuclease Free Water to elute our samples for highest compatibility with downstream PCR. For other applications replace with Buffer AVE.


Note
In this protocol we used 80 µl Nuclease Free Water to elute our samples. For other applications this volume could be adjusted. For highest yield a two-stage elution with 1/2 and 2/2 elution volume can be used, but was not tested in this protocol. Please see the Qiagen Viral RNA Mini Kit manual for more details when designing for your specific application.

Incubate Duration00:01:00 at TemperatureRoom temperature

1m
Place the entire QIAamp Mini Spin Column & tube into the portable centrifuge.
Close the lid of the device, check the red LED is illuminated, and push the front button beside the LED.
Wait Duration00:01:00 for the liquid to spin down.

Collect the labeled Amount1.5 mL tube.
Expected result
The Amount1.5 mL tube now contains ~50-60 µl of RNA sample in Nuclease Free Water. Please store appropriately (or move immediately to downstream testing.



Discard the QIAamp Mini Spin Column.
RT-PCR
RT-PCR
5m 53s
5m 53s
Thaw at TemperatureRoom temperature and place on ice:
  1. PCR Master Mix
  2. PCR primers
  3. Water
  4. RNA Sample

The PCR Mix used in this protocol:
ReagentApplied Biosystems™ TaqMan™ Fast Virus 1-Step Master Mix for qPCR Applied Biosystems (ThermoFisher Scientific)Catalog #44-444-32

Primers were purchased from Integrated DNA Technologies (IDT).
Setup the following reaction:

Component (Stock Concentration) Volume

PCR Master Mix (4X) Amount6.25 µL
Forward primer (10 µM) Amount1.50 µL
Reverse primer (10 µM) Amount1.50 µL
RNA Sample (variable) Amount10 µL
Nuclease Free Water Amount5.50 µL
Total Amount25 µL


Set-up the following program on the thermal cycler:

Step Temperature Time Cycles
Reverse Transcription Temperature55 °C Duration00:05:00 1
Heat Activation Temperature95 °C Duration00:00:20 2 1
Denaturation Temperature95 °C Duration00:00:03 45-60
Annealing & Extension Temperature60 °C Duration00:00:30 45-60
Hold Temperature4 °C
5m 53s
Protocol references