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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Related Experiment Video

Updated: Mar 2, 2026

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Published on: September 24, 2015

Immuno-PCR with digital readout.

Hendrik Schröder¹, Maximilian Grösche², Michael Adler¹

¹Chimera Biotec GmbH, Emil-Figge-Str. 76A, D-44227 Dortmund, Germany.

Biochemical and Biophysical Research Communications

|May 10, 2017

Summary

This summary is machine-generated.

Digital droplet PCR (ddPCR) enhances Immuno-PCR (IPCR) for highly sensitive protein biomarker detection. This ddIPCR method offers improved accuracy and precision over real-time PCR, demonstrating robust results for cytokine analysis.

Keywords:

Antibody Biomarker Digital droplet polymerase chain reaction Immuno PCR Immunoassays Ultra-sensitive

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Area of Science:

Biochemistry
Molecular Biology
Immunotechnology

Background:

Immuno-PCR (IPCR) integrates ELISA antigen detection with PCR amplification for sensitive target detection.
Real-time PCR (rtPCR) is commonly used for quantifying antigens in IPCR, correlating DNA amplification to target concentration.
Existing methods face limitations in accuracy and precision for certain biomarker quantification tasks.

Purpose of the Study:

To implement digital droplet PCR (ddPCR) for direct DNA copy quantification in Immuno-PCR assays.
To evaluate the sensitivity and robustness of ddIPCR for protein biomarker detection.
To compare the performance of ddIPCR with traditional rtPCR in an IPCR context.

Main Methods:

Developed and tested two IPCR formats: magnetic microbead-based and microplate-release.
Utilized digital droplet PCR (ddPCR) for direct quantification of amplified DNA markers.
Applied the optimized microplate-release ddIPCR assay to detect Interleukin-2 (IL2) and Interleukin-6 (IL6) biomarkers.

Main Results:

The microplate-release IPCR format demonstrated high robustness and sensitivity.
ddIPCR enabled direct correlation between signal response and analyte concentration, improving accuracy and precision.
The assay achieved low coefficients of variation: 5.0% for IL2 and 7.4% for IL6.

Conclusions:

Digital droplet PCR provides a significant advancement for Immuno-PCR, enhancing sensitivity and quantification accuracy.
The ddIPCR-based Immuno-PCR assay is a robust and precise method for detecting protein biomarkers like cytokines.
This technology holds promise for sensitive and reliable biomarker analysis in various research and diagnostic applications.