Construction of an Internal Control to Quantitate Multiple Porcine Cytokine mRNAs by RT-PCR | Zendy

N.R. Jayagopala Reddy | Zendy; B.N. Wilkie | Zendy; Bonnie A. Mallard | Zendy

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Construction of an Internal Control to Quantitate Multiple Porcine Cytokine mRNAs by RT-PCR

Author(s) -

N.R. Jayagopala Reddy,

B.N. Wilkie,

Bonnie A. Mallard

Publication year - 1996

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/96215st05

Subject(s) - oligonucleotide , reverse transcriptase , biology , primer extension , microbiology and biotechnology , primer (cosmetics) , polymerase chain reaction , reverse transcription polymerase chain reaction , overlap extension polymerase chain reaction , messenger rna , endonuclease , computational biology , dna , chemistry , genetics , gene , organic chemistry

A multiple internal control was constructed to be used as an exogenously added control in reverse transcription polymerase chain reaction (RT-PCR) for pig cytokines. It consists of 5' and 3' primer sequences in the order of beta 2 microglobulin (beta 2-m), IL-1, IL-4, IL-6, IL-8, IL-2, IL-10, TNF-alpha, TNF-beta and IFN-gamma. Construction was accomplished by overlapping and extension PCR (OE-PCR) utilizing short oligonucleotides. The primers were designed to give two products of different sizes on co-amplification of control and target RNAs by RT-PCR in a single tube. This permits analysis of message for several cytokines using a single exogenously added competitive template. Incorporated endonuclease sites allow construct modification by oligonucleotide addition.

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