Validation of a quantitative method for real time PCR kinetics

Liu, Weihong; Saint, David A.

doi:10.1016/s0006-291x(02)00478-3

Cited by 307 publications

(237 citation statements)

References 24 publications

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“…First-strand cDNA was synthesized using the energicScript ® cDNA synthesis kit (ShineGene, Co., Ltd., Shanghai, China) according to the manufacturer's protocol. RT-PCR was performed as described in a previous report (26). PCR primers and probes for murine GATA-3, Arg-1 and the housekeeping genes, such as GAPDH, were designed by ShineGene Co., Ltd., based on cDNA sequences obtained from the GenBank database (Table I).…”

Section: Methodsmentioning

confidence: 99%

Th2-like immune response in radiation-induced lung fibrosis

Hân

Zhang²,

Xie

et al. 2011

Oncol Rep

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Section: Methodsmentioning

confidence: 99%

Th2-like immune response in radiation-induced lung fibrosis

Hân

Zhang²,

Xie

et al. 2011

Oncol Rep

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“…The primer efficiency E was determined for each target sequence by calculation of the slope of the respective amplification curves as described. 36,37 ⌬Ct values were standardized against an internal GAPDH control, and fold increase was calculated as (1ϩE) -⌬ ⌬Ct , relative to an unstimulated control at each time point. 33,34 The following primers were used: A3B: 3B4 (GGTCAGCAAT-TCATGCCTTGGTAC, nt 574-597), 3B5 (CCCTG TAGATCTGGGCCGGGTCC, as, nt 750-728).…”

Section: Methodsmentioning

confidence: 99%

“…The real-time PCRs were normalized for the amplification efficiencies of each transcript and were normalized to that of GAPDH cDNA in each sample. 36,37 These calculations allowed us to directly compare the expression of the four APOBEC3 transcripts. 36,37 In unstimulated primary hepatocytes, A3B Fig.…”

Section: Expression Of Apobec3 Genes In Human Livermentioning

confidence: 99%

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication

et al. 2006

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Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-␣) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B L inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B S and A3C had no effect. A3B L and A3B S were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. T he hepatitis B virus (HBV) infects more than 350 million people worldwide and is a leading cause of end-stage liver disease and of hepatocellular carcinoma. 1 HBV is non-cytopathic for hepatocytes; however, most newly HBV infected adult patients develop acute hepatitis because of a strong immune response that clears HBV from the liver, whereas approximately 5% of newly HBV-infected adult patients generate insufficient immunity and become chronically infected. 1,2 Administration of interferon alpha (IFN-␣) is a mainstay of therapy for chronically HBV-infected patients. 2 Interferons restrict the replication of HBV by inducing the expression of antiviral proteins that inhibit the formation of replication-competent HBV nucleocapsids, and ultimately can result in the resolution of the chronic HBV infection. 2-7 HBV and other hepadnaviruses replicate their partially double-stranded DNA genome within cytoplasmic core particles by reverse transcription of encapsidated pregenomic RNA and thus are related to retroviruses. 8,9 The cytidine deaminase APOBEC3G (A3G), which is encoded within a cluster of seven related editing enzymes (APOBEC3A-G) on chromosome 22, provides broad innate immunity against exogenous and endogenous retroelements. 10-16 Encapsidated into the retroviral particle A3G deaminates dCs of the retroviral minus strand

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“…This method is a simplified approach for efficiency calculation as the efficiency may change as the starting copy concentration varies and therefore may not be accurate. 26,27 Nevertheless, the method is useful to compare relative amplification efficiencies. The acceptability of the relative efficiencies can be confirmed by demonstrating that when patient cDNA is diluted in a 10-fold series, the transcript values are not significantly different from the expected 1 log reduction at each dilution step.…”

Section: Appropriate Standards and The Influence Of Pcr Amplificationmentioning

confidence: 99%