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Sigma-Aldrich

 

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Article 6: qPCR data analysis 2 – Controls and Troubleshooting

12 December 2009 | By

The tremendous increase in the number of laboratories using qPCR and publications relying on qPCR data are testament to the rapid uptake of this technology. When preceded by reverse transcription (RT-qPCR) it is regarded as the reference technique for validation of previously derived data such as from microarray studies and…

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Article 5: qPCR data analysis – Amplification plots, Cq and normalisation

9 October 2009 | By

A pivotal attraction of qPCR technology is its apparent lack of complication; an assay consisting of the simple procedure of combining oligonucleotides, PCR mastermix buffer and nucleic acid template to produce a qPCR reaction is perceived as undemanding. This practical simplicity is complemented by the absence of any requirement for…

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Article 4: Optimisation of the PCR step of a qPCR assay

30 July 2009 | By

The deceptive simplicity of a typical qPCR assay is an important reason for the exponential growth in the adoption of qPCR-related technologies for both research and diagnostic applications. The only requirements for obtaining ostensibly quantitative data are a mixing of primers, DNA and a mastermix, their distribution into individual tubes…

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Article 3: qPCR Assay Design

29 May 2009 | By

Real-time PCR (qPCR) data are reliable only if they result from a robust qPCR assay that has been carefully designed, validated and optimised. This process requires an extensive assay design procedure aimed at generating an optimum primer/probe/amplicon combination to allow accurate quantification of nucleic acids with minimum need for post-PCR…

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Article 2: Reverse Transcription – a necessary evil

20 March 2009 | By

The fluorescence-based quantitative real-time polymerase chain reaction (qPCR)1-3, is the most widely used method to detect and measure minute amounts of DNA in a wide range of samples extracted from numerous sources. Since all currently available thermostable polymerases are DNA-dependent, RNA must be converted ("reverse transcribed") into DNA prior to…

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The importance of sample quality for qPCR

7 February 2009 | By Tania Nolan, Global Manager, Sigma-Aldrich and Stephen Bustin, Professor of Molecular Science, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry

The fluorescence-based quantitative real-time polymerase chain reaction (qPCR)1-3, has the ability to detect and measure minute amounts of DNA in a wide range of samples extracted from numerous sources. In combination with reverse transcription (RT), the use of this technology has revolutionised life sciences, agriculture and medical research4,5. In addition,…