Jan 30, 2026

Public workspace1 Step RT- QPCR 

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbo5enlpk/v1
  • Chelsie Steele1,
  • Yujie Fan1
  • 1Caltech
Chelsie Steele
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DOI: https://dx.doi.org/10.17504/protocols.io.81wgbo5enlpk/v1
Protocol Citation: Chelsie Steele, Yujie Fan 2026. 1 Step RT- QPCR . protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbo5enlpk/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: January 29, 2026
Last Modified: January 30, 2026
Protocol Integer ID: 241809
Keywords: step rt qpcr, qpcr, rt
Abstract
description of how to do a 1 step rt qpcr
Troubleshooting
Organoid RNA extraction
All RNA was extracted using the following protocol as described in our paper dx.doi.org/10.17504/protocols.io.81wgbr95qlpk/v1
1 Step RT-QPCR
  • Use RNase-free technique throughout.
  • Decide your multiplex groups (up to 4 targets per well, including GAPDH), making sure fluorophores do not overlap.
  • Thaw master mix, probes, and RNA on ice. Mix gently and quick-spin.
Measure RNA concentration for each sample.
Dilute each RNA sample with nuclease-free water so all samples have the same concentration (so that 2 µL gives the same RNA input for every sample)
Keep normalized RNA on ice
Create a plate map:
List each sample.
For each probe set/condition, assign 2 technical replicates per sample.
Total wells = samples × conditions × replicate
Label your 384-well plate orientation and corresponding wells
For each multiplex condition (each “probe set”), make a probe mix so pipetting is consistent.In a labeled tube for Probe Set 1, combine the probes for that multiplex (up to 4 probes).Repeat for Probe Set 2, 3, and 4
Make one “reaction mix” per probe set that contains everything except RNA. Per well (10 µL total):
  • 5.0 µL IDT PrimeTime One-Step RT-qPCR Master Mix
  • 0.5 µL of each probe (up to 4 probes total; 2.0 µL max probe volume)
  • Nuclease-free water to bring the non-RNA volume to 8.0 µL
  • RNA will be 2.0 µL added later
For each probe set, calculate number of reactions needed:
(number of wells for that probe set) × (1.1 or 1.2) to account for pipetting loss
In a chilled tube, combine for that probe set:
PrimeTime One-Step RT-qPCR Master Mix (5 µL × number of reactions)
Probe mix components (0.5 µL each probe × number of reactions)
Nuclease-free water to reach 8 µL per reaction
Mix gently by pipetting, then quick-spin.
Keep mixes on ice and protected from light.
Add 8.0 µL of the appropriate probe-set reaction mix into each assigned well of the 384-well plate.
Add 2.0 µL of the corresponding normalized RNA sample into each well.
For no-template controls (NTCs), add 2.0 µL nuclease-free water instead of RNA.
Seal the plate with an optical seal. Quick-spin the plate (brief spin) to remove bubbles and collect liquid at the bottom.
Load the plate into the Bio-Rad CFX Opus 384.
Set up the run as a one-step RT-qPCR multiplex assay:
  • Assign fluorophores/channels matching your probes.
  • Confirm plate type (384-well) and reaction volume (10 µL).
Use the cycling program recommended by IDT for PrimeTime One-Step RT-qPCR Master Mix
Start the run.
Analysis
After the run, verify amplification curves and baseline/threshold settings
Confirm NTCs show no amplification.
Export Cq values and analyze using your preferred method