Jul 03, 2025
Version 1
Automated high-throughput RT-PCR V.1
- Alex Veith1,
- Anna Shen1,
- Christopher Bradfield1
- 1University of Wisconsin-Madison
Protocol Citation: Alex Veith, Anna Shen, Christopher Bradfield 2025. Automated high-throughput RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dzm1vqe/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: February 12, 2025
Last Modified: July 03, 2025
Protocol Integer ID: 120124
Keywords: RT-PCR, Automated liquid handling, EpMotion, pcr this protocol, pcr, gene expression target, diluted rna, μl of diluted rna, different numbers of gene expression target, well pcr plate for efficiency, gene expression, rna, well pcr plate, custom rt, throughput rt, including primer, gene, technical replicate, primer, rt, step master mix
Funders Acknowledgements:
National Institute of Environmental Health Sciences
Grant ID: R35ES028377
Abstract
This protocol provides an outline for planning, setting up, and running an epMotion® M5073 for high-throughput RT-PCR. This protocol leverages a set of pre-written template programs to accommodate different numbers of gene expression targets. The template programs have been designed to dispense 2 μL of diluted RNA and 8 μL of one-step master mix (including primers and probes) to create 10 μL reactions on a 384-well PCR plate. Diluted RNA is to be arranged in a 96-well PCR plate for efficiency. We also provide the steps necessary to create custom RT-PCR runs for more advanced users. These steps also detail the settings used in our predefined programs. Users can expect consistent results with minimal variability between technical replicates that are quick and reliable.
Attachments
Guidelines
The template protocols were optimized using RNA diluted in water (50 ng/μL), Taqman™ Gene Expression Assays, and Promega GoTaq® Probe One-Step Master Mix, and then run on a QuantStudio™. Templates must be edited if changes are made to consumables, reagents, or the protocol. Please also validate the data conversion template. Post-run log files can also be obtained with detailed run information.
Materials
Consumables
Eppendorf twin.tec® PCR PlatesEppendorfCatalog # 951020745 (any color)
Microcentrifuge tubesEppendorfCatalog #022364111
96 Well PCR Plates, Semi-SkirtedPhenix ResearchCatalog #1147B52
Only if using multichannel master mix dispensing
epMotion Reservoir 10 mLEppendorfCatalog #0030126521 (may need other sizes)
Reagents
Diluted RNA (we use 50ng/
GoTaq® Probe RT-qPCR KitPromegaCatalog #A6121 (or equivalent)
TaqMan™ Gene Expression AssayThermo Fisher Scientific
Protocol materials
GoTaq® Probe RT-qPCR KitPromegaCatalog #A6121
TaqMan™ Gene Expression AssayThermo Fisher Scientific
Eppendorf twin.tec® PCR PlatesEppendorfCatalog # 951020745
Microcentrifuge tubesEppendorfCatalog #022364111
96 Well PCR Plates, Semi-SkirtedPhenix ResearchCatalog #1147B52
epMotion Reservoir 10 mLEppendorfCatalog #0030126521
epMotion® reservoir 10mLEppendorfCatalog #0030126521
Troubleshooting
Safety warnings
The epMotion® does have optical sensing. However, this feature is not used in any volume detection steps. Therefore, you must be accurate and precise with your volumes.
Before start
Screenshots and equipment notes have been embedded into the notes for several steps.
Select the protocol depending on whether the experiment can utilize a pre-defined template program (recommended) or whether a custom program is needed. This protocol provides a basic outline for creating a custom program; however, additional training may be required.
Predefined RT-PCR Template Programs38 steps
Follow this protocol to use the predefined template programs. This is recommended for most users.
Simulation runs for each template program are attached.
Experimental Design
Each epBlue® protocol has been written based on the number of gene expression targets per plate (column A). Based on the number of probes and technical duplication, we have also listed the maximum number of samples that can be included on a single 384-well RT-PCR plate.
| # of Probes | Maximum Sample # | |
| 1 | 192 | |
| 2 | 96 | |
| 3 | 64 | |
| 4 | 48 | |
| 5 | 32 | |
| 6 | 32 | |
| 7 | 24 | |
| 8 | 24 |
The pre-designed protocols have been written to perform one-step RT-PCR reactions. In these protocols, 2µL diluted RNA is mixed with 8µL master mix containing all other required reaction components for a 10µL reaction.
Estimate Minimum RNA Volume
The 96 Well PCR Plates, Semi-SkirtedPhenix ResearchCatalog #1147B52 have a "dead volume" of 8µL with the epMotion®. Use this formula to estimate the volume of RNA required for your experiment. RNA concentration is up to the user.
Estimated Minimum RNA Volume Required = (volume RNA/well [2] * technical replicates [2] * # of gene expression assays) + 8µL
Note
The estimate for the RNA volume is reliable, but it will need to be verified on the epBlue® software.
RNA samples will be aliquoted into an 96 Well PCR Plates, Semi-SkirtedPhenix ResearchCatalog #1147B52 according to the layout below (Figure 1). Aliquot the volumes determined by the prior calculation. Each well will likely have an equal volume. The layout of the 384-well reaction plate is found at .
Figure 1. Plate layout.
Order RNA samples in ascending order as indicated by this layout.
Note
Ensure diluted RNA is centrifuged to eliminate splatter and bubbles
Select protocol format based on the user's experimental design. The default works well for low to moderate sample sizes (8-48), while the high sample format works better for experiments with a high N (>48) and a low number of gene expression targets (1-4).
Single-channel Master Mix Protocols33 steps
This is for protocols utilizing the single-channel pipette head to dispense the master mix. This is the default and is useful for multiple gene expression targets (2-8) and small sample sizes.
Estimate Minimum Master Mix Volume (per gene expression target)
The Microcentrifuge tubesEppendorfCatalog #022364111 have a "dead volume" of 4µL. Use this formula to estimate the volume of Master Mix required for your experiment. This is the volume needed for each gene expression assay.
epMotion® Volume: (number of samples * volume/well [8] * technical replicates [2]) + 4µL
Estimated Minimum Volume Required = epMotion® Volume + (0.1 * epMotion® Volume)
Note
The estimate for the Master Mix volume is reliable, but it will need to be verified on the epBlue® software.
Sample and Master Mix Prep
Aliquot RNA with the minimum calculated volume into 96 Well PCR Plates, Semi-SkirtedPhenix ResearchCatalog #1147B52 per the format described in Figure 1 .
Note
Excess volume can be prepared if desired. We often aliquot excess so that one RNA plate can be reused for multiple gene expression experiments. We have also stored RNA in sealed plates at -80°C. Regardless of the volume aliquoted, it is necessary to be accurate and record the volume for the epBlue® software.
Prepare Master Mix(s) for the number of gene expression assays used in this experiment (1-8).
epBlue® Login and Program Selection
Log in to the epBlue® software using the guest login
user: RT_PCR_Guest
pass: guest
Click "Application Runner."
Note
Screenshot
Select the program that corresponds to the number of gene expression targets to be run on a single 384-well plate.
Note
Screenshot
Note
The programs listed here use a single-channel pipette head to dispense master mix. A separate folder for programs uses a multichannel pipette head to dispense master mix. These programs are only recommended if you have a large number of samples (>48). The larger number of samples limits the number of targets that can be included on a single plate. and select Large Sample N if your experimental design requires.
Users are first prompted to start either a simulation or an actual run. Ensure the machine ID is selected (blue) to begin a live run. This page details the worktable layout and the tools needed for this protocol (single and multi-channel P50 pipette heads). Then click the next button to proceed.
Note
Screenshot
Users are then prompted to set up other basic parameters for each run. Please ensure that the following settings (red arrows) are selected (blue).
- Input volumes manually
- Detect tips
- Check labware placement
- Check tube lid removed
Note
Screenshot
Input the number of samples. The program will have a maximum number.
The next page shows the worktable set up. This is the same for every program, and it will be described in a future step. Click Next at the bottom of the screen.
RNA Volume Validation
Verify diluted RNA volume. The program will give you the minimum volume required for the run (red arrow). As each well will likely have equal volumes, select the "Set all volumes" button to quickly assign a volume to each well. This will include volumes in unused wells, but this is irrelevant.
Input diluted RNA volumes. Volumes must be accurate. Click next to proceed.
Master Mix Volume Validation
The next page indicates the master mix volume required for each gene expression assay. The software indicates the expected amount of master mix (red arrow). In our experience, this protocol runs optimally when at least 10% extra master mix is prepared and is reflected in the estimate formula ( )
Note
Screenshot
Input diluted Master Mix volumes. Volumes must be accurate. Click next to proceed.
Worktable Setup
Worktable positions used. Empty waste bin before use.
Note
Screenshot
Ensure that the TS-50 and TM-50 pipette heads are in positions T1 and T2. It does matter which spot each pipette head is in.
Note
Equipment Information:
Equipment
TS 50 single-channel dispensing tool
NAME
epMotion® Dispensing tools
TYPE
Eppendorf
BRAND
Catalog no. 960001010
SKU
Equipment
TM 50-8 eight-channel dispensing tool
NAME
epMotion® Dispensing tools
TYPE
Eppendorf
BRAND
Catalog No. 960001044
SKU
Insert a 384-well plate into the TMX slot. Press gently but ensure theplate is secure.
Eppendorf twin.tec® PCR PlatesEppendorfCatalog # 951020745
Place 96-well thermoadapter in slot B1.
Note
Equipment Information:
Equipment
Thermoadapter for PCR
NAME
epMotion Thermoracks and Plate Accessories
TYPE
Eppendorf
BRAND
Catalog No. 960002199
SKU
Place a 96-well PCR plate onto the thermoadapter already in position B1.
96 Well PCR Plates, Semi-SkirtedPhenix ResearchCatalog #1147B52
Add tips to the worktable at position A2. These programs all use PCR clean p50 filter tips. The tip rack has marks on the side that indicate to the machine what tips are present in the box. When placed onto the worktable, those marks need to be on the left.
Note
If the program requires more than 96 tips, the software will stop at that point and allow users to add more tips.
Note
Equipment Information:
Equipment
epT.I.P.S.® Motion Pipette Tips
NAME
epMotion Pipette Tips
TYPE
Eppendorf
BRAND
Catalog No. 0030014413
SKU
P50
SPECIFICATIONS
Place ReservoirRack in position B2.
Note
Equipment Information:
Equipment
ReservoirRack
NAME
epMotion® ReservoirRacks and Modules
TYPE
Eppendorf
BRAND
Catalog No. 960002148
SKU
Place the ReservoirRack Module PCR into the ReservoirRack. This module occupies six of the seven slots. The gap should be on the left side (position 1).
Note
Equipment Information:
Equipment
ReservoirRack Module PCR
NAME
epMotion® ReservoirRacks and Modules
TYPE
Eppendorf
BRAND
Catalog no. 5075751933
SKU
Place master mix tubes into designated slots (below) and input volumes into the software.
Input volume. The "Set all volumes" control does not work well for this step.
| Probe # | 1 Target Program | 2 Target Program | 3 Target Program | 4 Target Program | 5 Target Program | 6 Target Program | 7 Target Program | 8 Target Program | |
| 1 | 2_2_A1 | 2_2_A1 | 2_2_A1 | 2_2_A1 | 2_2_A1 | 2_2_A1 | 2_2_A1 | 2_2_A1 | |
| 2 | 2_2_B1 | 2_2_B1 | 2_2_B1 | 2_2_B1 | 2_2_B1 | 2_2_B1 | 2_2_B1 | ||
| 3 | 2_2_C1 | 2_2_C1 | 2_2_C1 | 2_2_C1 | 2_2_C1 | 2_2_C1 | |||
| 4 | 2_2_D1 | 2_2_D1 | 2_2_D1 | 2_2_D1 | 2_2_D1 | ||||
| 5 | 2_2_E1 | 2_2_E1 | 2_2_E1 | 2_2_E1 | |||||
| 6 | 2_2_F1 | 2_2_F1 | 2_2_F1 | ||||||
| 7 | 2_2_G1 | 2_2_G1 | |||||||
| 8 | 2_4_A5 |
ReservoirRack Module PCR Master Mix Positions
Number indicates target order and position in the ReservoirRack Module PCR
Run
Click "Run." Users may wish to go and set up the RT-PCR machine with the experimental layout (see )
Note
- During the run, users can pause and abort the run if they identify an issue.
- If the run is aborted, users will need to remove any pipette heads from the tool positions (next to TMX) so that the machine can return an attached pipette head.
- The software will analyze the run parameters and notify users of any potential issues (such as not enough tips). Should the tips run out during the run, the machine will stop and allow users to exchange tip boxes.
Click "OK" on the pop-up declaring "The application ended successfully".
Click "Exit."
Open the door, remove the 384-well plate, and seal.
RT-PCR
1000 rpm, 00:05:00 , 384-well plate, tabletop centrifuge
Set up RT-PCR machine plate layout.
Figure 2. 384-well Plate Pattern.
Gene expression targets are ordered left to right and indicated by different-colored wells. White and yellow text indicate the technical duplicates for each sample and target.
Run the plate on QuantStudio™.
Note
Preparing the QuantStudio™ is not a part of this protocol.
Data Analysis
Export results as a CSV. Select all fields in the Results tab. This will make copying and pasting data in the next step easier.
Copy the results to an Excel template file. This template converts the exported long data format to a plate layout and duplicate format. Make sure the data is pasted into the template correctly.
ExpressionStudioConverter.xltx43KB (newer software, uses Cq)
RT-PCR_DataConverter.xltx41KB (for older software, uses Ct)Exit to the Start Screen, go to the log viewer, and retrieve the PDF printout of the log file for the run.
Acknowledgements
We want to acknowledge Patrick Carney, Ksenija Korac, and Manabu Nukaya of the Sean Ronnekleiv-Kelly Lab for their assistance in validating our test protocols.