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Related Concept Videos

Real Time RT-PCR02:57

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...
PCR01:32

PCR

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Related Experiment Video

Updated: Jul 11, 2026

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
04:52

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Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance.

Ricky R Lareu1, Karthik S Harve, Michael Raghunath

  • 1Tissue Modulation Laboratory, Division of Bioengineering, Faculty of Engineering, National University of Singapore, Division Office Block E3A #04-15, 7 Engineering Drive 1, Singapore 117574, Singapore.

Biochemical and Biophysical Research Communications
|September 15, 2007
PubMed
Summary

Adding inert macromolecules to polymerase chain reaction (PCR) and reverse transcription (RT) assays dramatically improves sensitivity and efficiency. This biomolecular crowding technique better mimics intracellular conditions for enhanced DNA amplification.

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Last Updated: Jul 11, 2026

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
04:52

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Published on: July 11, 2011

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp
10:44

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Rapid PCR Thermocycling using Microscale Thermal Convection
09:02

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Published on: March 5, 2011

Area of Science:

  • Molecular Biology
  • Biophysics

Background:

  • Polymerase chain reaction (PCR) and reverse transcription (RT) are vital molecular biology techniques.
  • Current in vitro PCR/RT methods do not replicate the crowded intracellular environment where DNA polymerases naturally function.

Purpose of the Study:

  • To investigate the impact of macromolecular crowding on in vitro PCR and RT reactions.
  • To enhance the efficiency, sensitivity, and stability of PCR and RT assays.

Main Methods:

  • Inert macromolecules were added to in vitro reverse transcription (RT) and polymerase chain reaction (PCR) assays.
  • The effects of macromolecular crowding on reaction kinetics, sensitivity, yield, specificity, and enzyme stability were evaluated.

Main Results:

  • Macromolecular crowding significantly enhanced PCR and RT parameters, including 8- to 10-fold increased sensitivity.
  • Improvements were observed in polymerase processivity, specific amplicon yield, primer annealing, and specificity.
  • Enhanced DNA polymerase thermal stability and faster reaction kinetics were achieved.

Conclusions:

  • Macromolecular crowding is a critical biophysical parameter that can be leveraged to optimize in vitro DNA amplification techniques.
  • This approach offers a more biologically relevant method for improving PCR and RT assays for diverse applications.