ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data

Perkins, James R.; Dawes, J. D. K.; McMahon, Stephen B.; Bennett, David L.H.; Orengo, Christine A.; Kohl, Matthias

doi:10.1186/1471-2164-13-296

Cited by 204 publications

(155 citation statements)

References 25 publications

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“…In addition to microarray-based studies on the expression pro ling, other methods have been developed for the detection of miRNAs, such as quantitative real-time PCR [19]. In order to investigate the involvement of miR-222 in tumorigenesis of CC, RT-PCR was done to detect the miR-222 expression in normal cervical tissues and in CC.…”

Section: Discussionmentioning

confidence: 99%

MicroRNA-222 promotes the proliferation and migration of cervical cancer cells

Sun¹,

Zhang²,

Cheng³

et al. 2014

CIM

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Purpose:is study aimed to investigate the role of small non-coding in the development of cervical cancer (CC).Methods: Normal and CC specimens were obtained from 18 patients. HeLa and SiHa cells were grown in Dulbecco's modi ed Eagle's medium. RT-PCR, Western blot, migration assay, ow cytometry and immuno uorescence microscopy were used for analyses.Results: When compared with normal cervical tissues, miR-222 was upregulated in human CC, and the extent of up-regulation was associated with the extent and depth of CC invasion. Expression of miR-222 was inversely related to the expression of phosphatase and tensin homolog (PTEN) and p27. e reduced the expression of PTEN and p27 by miR-222 in HeLa cells and SiHa cells was associated with increased proliferation and migration of CC cells. e expression of proteins (E-cadherin and paxillin) related to the proliferation and migration was also elevated.Conclusion: MiR-222 plays an important role in the tumorigenesis of CC, possibly by speci cally down-regulating p27 Kip1 and PTEN. Our ndings suggest that miR-222 may serve as a new therapeutic target in CC.

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Section: Discussionmentioning

confidence: 99%

MicroRNA-222 promotes the proliferation and migration of cervical cancer cells

Sun¹,

Zhang²,

Cheng³

et al. 2014

CIM

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“…Data were exported from MxPro software (Agilent), imported into R, and analyzed using ReadqPCR and NormqPCR (Perkins et al 2012;R Core Team 2017). Potential normalizers vcl, gstd2, flna, and rps20 were evaluated for stability using geNORM (Vandesompele et al 2002) implemented within NormqPCR; the geometric mean of the two normalizers with the lowest M-value was used to normalize samples.…”

Section: Salmon Lice Rt-qpcr Validationmentioning

confidence: 99%

Host–parasite transcriptomics during immunostimulant-enhanced rejection of salmon lice (Lepeophtheirus salmonis) by Atlantic salmon (Salmo salar)

Sutherland

Covello

Friend

et al. 2017

FACETS

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Salmon lice (Lepeophtheirus salmonis) are important ectoparasites of wild and farmed salmonids and cause major losses to the salmon farming industry throughout the Northern Hemisphere. With the emergence of resistance to several commonly used parasiticides, novel control strategies and integration of multiple treatment options are needed, including host immunostimulation. Here, we investigate the effects of a functional feed containing a peptidoglycan and nucleotide formulation on L. salmonis infection of Atlantic salmon (Salmo salar) by characterizing lice infection levels, the expression of several host immune genes, and the parasite transcriptomic response to the immunostimulated host. Although initial infection intensities were low, the low dose (LD) immunostimulant diet reduced the total lice burden by 50% relative to controls. Immunostimulant fed hosts upregulated interleukin-1β in the skin and spleen. This gene has been implicated in successful responses of several salmonid species to salmon lice but is typically not observed in Atlantic salmon, suggesting a favorable influence on the immune response. Lice infecting LD immunostimulated salmon overexpressed genes putatively involved in parasite immunity, including carboxylesterases, and underexpressed genes putatively involved in feeding (e.g., proteases). These lice response genes further improve the characterization of the transcriptome of the non-model parasite by identifying genes potentially involved in evading host immunity.

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“…For calculations by BestKeeper, delta Ct method and RefFinder, the average Ct value was used directly. All calculations, except the ones done by the web based RefFinder, were carried out in R version 3.3.2 (R Core Team 2016) with "NormqPCR" package (Perkins et al, 2012). geNorm calculates the expression stability value M by assessing the mean pairwise expression ratio for each candidate gene against all the other candidates (Vandesompele et al, 2002).…”

Section: Determination Of Reference Gene Expression Stabilitymentioning

confidence: 99%

Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae)

Nagy

Németh

Juhász

et al. 2017

Preprint

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Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors in question by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples of Lethrus apterus, an earth-boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. These reference genes can be used to study the hormonal regulation of reproduction and parental care in Lethrus apterus in the future. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples of Lethrus apterus, an earth boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. These reference genes can be used to study the hormonal regulation of reproduction and parental care in Lethrus apterus in the future.

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ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data

Cited by 204 publications

References 25 publications

MicroRNA-222 promotes the proliferation and migration of cervical cancer cells

MicroRNA-222 promotes the proliferation and migration of cervical cancer cells

Host–parasite transcriptomics during immunostimulant-enhanced rejection of salmon lice (Lepeophtheirus salmonis) by Atlantic salmon (Salmo salar)

Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae)

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