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

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Analysis of Small RNAs by Quantitative Reverse Transcription PCR

(Protocol summary only for purposes of this preview site)

Northern hybridization reveals the expression level and at same time the size(s) of a small RNA; it is the gold standard for small RNA validation and quantification. However, northern hybridization cannot detect low-abundance small RNAs, nor can it survey large numbers of small RNA species. In contrast, quantitative RT-PCR is more sensitive and can be adapted for high throughput (Fig.1). This protocol is adapted from Applied Biosystems TaqMan Small RNA Assays (Life Technologies Corporation 2011). TaqMan small RNA assays are designed to quantify RNAs 17200 nucleotides long, including siRNAs and miRNAs. TaqMan small RNA assays rely on a validated set of oligonucleotides comprising a stemlooped RT primer, a pair of PCR primers, and a TaqMan probe. Additional information on quantification of RNA by quantitative RT-PCR can be found in Chapter 9.


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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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