Real-Time PCR (qPCR) Learning Center

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Real-time PCR has become the tool of choice for its rapid and sensitive detection of nucleic acids across a wide dynamic range, from biological samples. Real-time PCR can be qualitative (determining the presence or absence of a gene sequence) or quantitative (copy number variation of an expressed gene). Real-time PCR that is quantitative, or qPCR, is very popular for analysis of gene expression.

Mastery of the qPCR application requires understanding of the main steps of the qPCR workflow.

This learning center is your guide to the theory and implementation of the qPCR technique as it is used in gene expression analysis, beginning with RNA isolation, through reverse transcription, to analysis of the qPCR reaction.

  • RNA Isolation

    RNA Isolation

    Cells containing your RNA of interest are lysed followed by RNA extraction to isolate RNA for gene expression. Careful sample handling, as well as RNAse inactivation and RNAse free solutions and consumables, is essential to ensure RNA integrity.

  • Reverse Transcription (RT)

    Reverse Transcription (RT)

    Reverse transcription is a process by which enzymes catalyze the transcription of RNA into a complementary DNA (cDNA) using reverse transcriptase. The cDNA in turn is the template for PCR amplification, followed by detection.

  • qPCR Assay Development

    qPCR Assay Development

    A successful qPCR assay requires efficient and specific amplification of the RNA transcript. The choice of target sequence, plex level, and primer design is significant, as they can affect amplification efficiency and specificity and thus the accuracy of qPCR assays.

  • qPCR Experiment/Amplification

    qPCR Experiment/Amplification

    In RT-qPCR, the amplification reaction is set up with PCR reagents, primers and probes in a master mix, and run using real time PCR instruments. The target specific fluorescence from the amplification reaction is monitored in real time and is proportional to the amount of amplified product.

  • qPCR Analysis

    qPCR Analysis

    Quantifying the absolute amount of a target sequence or comparing relative amounts of a target sequence between samples uses the Cq value and is calculated by your cycler's proprietary software. This value determines the PCR cycle number at which your sample’s reaction curve intersects the threshold line and provides a real signal. Real Time PCR has a reaction curve for each sample and therefore many Cq values.

GXB-Gene-Expression-Uni

More Quantitative PCR

Gene Expression University

Series 1: Courses Designed to Make You a qPCR Expert
Monday, April 4th – Friday, April 8th
One session per day starting at 9:00AM PDT/ 5:00PM BST

Join us for in‑depth technical courses in RT‑qPCR. This 5 part webinar series will give you an understanding of basic RT‑qPCR techniques and troubleshooting at each step of the gene expression workflow. Each class will include 45 minutes of instruction and 15 minutes of live Q&A. Slides and downloadable resources are available for each class.

On-Demand

CFX Opus Cloud Connectivity: BR.io

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The CFX Opus System seamlessly integrates with Bio-Rad's new cloud platform, BR.io, enabling you to get the most out of your instrument and minimize time at the bench. BR.io can be accessed from any internet connection using a Safari or Chrome web browser, and there is no software installation required.

BR.io cloud connectivity eliminates the need for a dedicated computer connected to the instrument and provides new capabilities.

 
  • Figure performing a remote setup

    Remotely set up CFX Opus runs, and start directly from the instrument

  • Instruments connected the cloud

    Monitor instruments and run progress remotely

  • Automatically transfer and store data in the cloud

  • Figure analyzing data

    Access and analyze data from anywhere