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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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Updated: Jul 28, 2025

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Published on: September 24, 2015

基于凝囊的数字定量聚合酶连锁反应.

Zheng Lin Tan¹, Masato Yasuura², Yukichi Horiguchi²

¹Sensing System Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan. tan.zhenglin@aist.go.jp.

Mikrochimica acta

|June 1, 2023

概括

基于凝囊的数字PCR (gc-dPCR) 提供了比传统的滴滴数字PCR (ddPCR) 更好的稳定性和灵敏性. 这种新的方法增强了核酸量化,降低了成本,使其更容易获得.

关键词:

滴滴数字聚合酶连锁反应凝囊数字聚合酶连锁反应;光微板阅读器;水凝是一种水凝.核酸量化的核酸量化方法

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Last Updated: Jul 28, 2025

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科学领域:

分子生物学分子生物学
生物技术是生物技术.
分析化学分析化学

背景情况:

滴滴数字PCR (ddPCR) 提供了高精度的核酸量化由于其线性和灵敏度.
在热循环过程中滴滴的稳定性是ddPCR的一个限制,影响量化的准确性.
目前用于提高滴滴稳定的方法,如化油或表面活性剂,增加成本并复杂化PCR后分析.

研究的目的:

引入一种基于凝囊的新型数字PCR (gc-dPCR) 方法.
提高数字PCR的稳定性和灵敏性.
为了减少与ddPCR相关的成本和复杂性.

主要方法:

开发水凝囊,以封装PCR反应混合物.
在这些水凝囊中进行PCR反应.
使用定量PCR (qPCR) 系统或光微板阅读器读取结果,与状ddPCR进行比较.

主要成果:

基于凝囊的数字PCR (gc-dPCR) 与状ddPCR相比显示出更高的光强度,表明灵敏度增加.
水凝囊在热循环过程中表现出优越的稳定性,而不是以油为基础的液滴.
该gc-dPCR方法允许对所有分区进行量化,防止低度样本的信息丢失.

结论:

基于凝囊的数字PCR (gc-dPCR) 通过提高滴滴稳定性和测试灵敏度,显著改善了滴滴数字PCR (ddPCR).
这种方法降低了数字PCR的整体成本,解决了在资源有限的环境中采用其的主要障碍.
该gc-dPCR方法可适应其他基于滴滴的PCR技术,在核酸量化中具有广泛的适用性.