Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Quantification of RNA by Real-Time RT-PCR

(Protocol summary only for purposes of this preview site)

The following protocol describes a real-time RT-PCR assay, using the two-enzyme, two-tube approach (see the section selecting the Enzyme(s) in the chapter introduction), carried out using either SYBR Green I or TaqMan chemistries. The protocol uses a PCR, volume of 20 L (although most manufacturers recommend 50-L reactions). However, if the PCR target is not very abundant (i.e., present at one to 10 copies per sample), a larger volume may yield better reproducibility between samples. For additional information on preparing high-quality RNA, choosing a priming method, and selecting an endogenous reference gene, see the section Performing Real-Time PCR in the chapter introduction.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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