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Quantifying Nucleic Acids by Real-Time PCR

Patricia de Winter

Genomics Research, Molecular Biology

This workshop is aimed at those who would like to use real-time PCR to quantify DNA or mRNA. Course length 8hrs

Product description

This workshop is aimed at those who would like to use real-time PCR to quantify DNA or mRNA. It is based upon previous workshops we have run over the past nine years for the BBSRC Molecular Training for Industry programme, King's College graduate school and The Physiological Society. Participants are expected to have a theoretical understanding of the polymerase chain reaction and reverse transcription. No prior knowledge of real-time PCR is expected.

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Part 1 - Sample Preparation and Quality Control

Introduction to Real-time PCR

A brief introduction to PCR and quantitative PCR is given, with details given about the origin and components of qPCR. Both it's importance in today's research environment and it's potential importance moving forward is discussed. Why conventional PCR is intrinsically non-quantitative and how qPCR overcomes this is explained, and the aims and objectives of this course are described.

Module 1: Sample/Template Preparation

The theory part of this module will take you through the isolation of total RNA and mRNA using a variety of different methods. We explore the different lysis buffers available, such as phenol-based or phenol-free, and the advantages and disadvantages of various homogenisation methods, such as sonication, grinding in liquid nitrogen and bead-mill homogenisers.

Finally we discuss the storage of RNA and how to reduce contamination of RNA with genomic DNA (gDNA).

Sample/Template Preparation Practical

In this section we demonstrate how to homogenise and purify RNA from tissue sample using a TissueLyser bead-mill homogeniser and spin columns. At the end of the practical we will have purified RNA ready for quality control analysis.

Module 2: Assessing Nucleic Acid Quantity, Purity and Integrity

This first part of this module details the two main methods available for assessing RNA/DNA quantity and purity (Spectrophotometry and Fluorimetry), the advantages and disadvantages of each technique and how to analyse and interpret the data produced.

Spectrophotometry Practical

Spectrophotometry can be used to provide information on the quantity and purity of your RNA sample. In this demonstration we use a NanoDrop spectrophotometer, taking you through how to quantify samples and also evaluate the RNA spectral data produced.

Fluorimetry Practical

A demonstration of how to use a Qubit fluorimeter to assess RNA quantity. We also discuss which type of assay to select for your sample, calibrating the instrument and how to analyse the data that are produced

Assessing Nucleic Acid Integrity

This lecture takes you through methods for analysing the integrity of the RNA sample to determine the extent of any degradation. This is very important as the integrity of RNA impacts heavily on many processes downstream.

We discuss the three main techniques available for assessing RNA integrity including the advantages and disadvantages of each one.

Microfluidic Capillary Electrophoresis Practical

This practical demonstrates the use of an Agilent Bioanalyzer microfluidic capillary electrophoresis instrument to assess the integrity of the RNA samples we isolated earlier.

Microfluidic Capillary Electrophoresis Data Analysis

This section shows you how to collect and interpret the data gained from your Agilent Bioanalyzer instrument to determine RNA sample integrity.

Module 3: Reverse Transcription (RT) of mRNA

In this lecture we talk about how to synthesise complementary DNA (cDNA) using a reverse transcription reaction (RT). We discuss the different RT priming strategies and enzymes that you can use for the reaction, as well as appropriate reaction conditions and RT efficiency and variability.

Reverse Transcription (RT) of mRNA Practical

In this section we show you how to set up an RT reaction with the total RNA isolated earlier. We will reverse transcribe each RNA sample in duplicate to see whether there is any variability in the RT.

Part 2 - qPCR Quantification of Nucleic Acids

Module 4: Assay Quality Control - Design and Specificity

This module takes you through the two main fluorescence detection strategies for determining the accumulation of product during qPCR. These two methods, intercalating dyes and sequence-specific probes, are then compared and the advantages and disadvantages of each discussed.
In addition, the module covers how you can assess your assay specificity using melt curve and agarose gel analysis. Finally, it takes you through assay design, specifically primer design, using web tools and software that are freely available.

Assay Quality Control - Primer Design Demonstration

This section demonstrates how to design primers for your qPCR experiment using an example RNA sequence. We use a number of web-based applications to assess primer suitability, such as Primer-BLAST, mFold and PCR Primer Stats.
http://www.ncbi.nlm.nih/gov/nucleotide

Assay Quality Control - cDNA Amplification Practical

How to perform an amplification reaction using a small amount of the cDNA prepared earlier to ascertain whether the primers you have designed are specific.

Assay Quality Control - Agarose Gel Analysis Practical

How to perform agarose gel analysis to check that a single product of the expected size is produced. We use a Bio-Rad E-gel electrophoresis system and a pre cast gel.

Module 5: qPCR using SYBR Green and Hydrolysis Probe Assays

This module discusses qPCR using known copy number standards, accuracy and repeatability, qPCR reliability and sampling dilute solutions.

qPCR using SYBR Green and Hydrolysis Probe Assays Practical

This section demonstrates how to prepare two qPCR runs with no-template controls, standards and cDNA samples of unknown copy number using both SYBR green and hydrolysis probe detection strategies.

qPCR using SYBR Green and Hydrolysis Probe Assays - Rotor-Gene qPCR Instrument

This section demonstrates how to use a Qiagen Rotor-Gene instrument to amplify your cDNA samples of unknown copy number.

qPCR using SYBR Green and Hydrolysis Probe Assays - Data Analysis

This section demonstrates how to analyse the data from your SYBR green and hydrolysis probe assays.

Module 6: MIQE Guidelines and Transparent Data Reporting

This section discusses the standards to adhere to when publishing your qPCR data, more specifically the Minimum Information for the Publication of Quantitative real-time PCR Experiments (MIQE) guidelines. In addition it discusses transparent data reporting.

Module 7: Assay Efficiency

This module explains qPCR assay efficiency and its importance in the quantification of qPCR data. It takes you through the methods for determining assay efficiency, it's effect on copy number and the variables that can influence efficiency.
In addition we show how to use direct methods to determine overall assay efficiency, as well as the use of indirect methods, such as mathematical models, to determine efficiency from individual amplification curves.

Calculating Assay Efficiency using LinRegPCR

In this section we discuss how to use LinRegPCR to calculate assay efficiency. Includes a practical demonstration of using LinRegPCR.