Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus

Strübe, Christina; Buschbaum, Sandra; Wolken, Sonja; Schnieder, Thomas

doi:10.1016/j.gene.2008.08.001

Cited by 55 publications

(39 citation statements)

References 21 publications

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“…According to Strube et al (2008), the housekeeping genes β-tubulin and ef-1α were chosen to correct for variations of mRNA amounts and cDNA synthesis efficiency. Primers and TaqMan™ minor groove binder (MGB) probes were designed using the Primer Express software (Applied Biosystems, Darmstadt, Germany).…”

Section: Plasmid Standardsmentioning

confidence: 99%

Molecular characterization and real-time PCR transcriptional analysis of Dictyocaulus viviparus major sperm proteins

2008

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Major sperm proteins (MSPs) represent a protein family occurring in nematodes only. Identification of the 3' and 5' untranslated region (UTR) completed the so far partial msp complementary DNA sequences of the bovine lungworm Dictyocaulus viviparus. The full-length transcript contains sequence tracts consistent with the Kozak and polyadenylation consensus sequence. On genomic level, three full-length sequences differing in three nucleotides were determined containing a 65-bp phase zero intron. Conceptual translation inferred two MSP isoforms due to one substitution within the 126-amino acid polypeptide. Bioinformatic analysis predicted that bovine lungworm MSP folds into an immunoglobulin-like seven-stranded beta sandwich as known for Caenorhabditis elegans and Ascaris suum. Furthermore, bovine lungworm MSP is confidentially predicted to be N-terminal-acetylated and secreted via a non-classical pathway. Quantitative real-time polymerase chain reaction analysis using ten developmental lungworm stages showed that msp is transcribed mainly in adult male parasites and in some degree in hypobiotic L5. However, marginal msp transcription was detectable in all of the investigated developmental lungworm stages.

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Section: Plasmid Standardsmentioning

confidence: 99%

Molecular characterization and real-time PCR transcriptional analysis of Dictyocaulus viviparus major sperm proteins

2008

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“…Plasmid standards containing msp and the evaluated reference genes elongation factor 1α (ef-1α) and β-tubulin (Strube et al 2008) as inserts were generated as described by Strube et al (2008Strube et al ( , 2009. To prepare the vit plasmid standard, the primers vit_for 5′-ACGCCGAT GATGGAGTA-3′ and vit_rev 5′-AGCCACTAAGGTTGA GAATGT-3′ based on the GenBank accession JN133288 were used.…”

Section: Poly(a) + Rna Isolation and First-strand Cdna Synthesismentioning

confidence: 99%

“…Primer and corresponding TaqMan™ minor groove binder (MGB) probes detecting msp, ef-1α, and β-tubulin transcripts were used as described by Strube et al (2008Strube et al ( , 2009. For vit detection, these oligonucleotides were designed with the AlleleID® software (version 7.01, PREMIER Biosoft International) based on GenBank accession no.…”

Section: Qpcr Runsmentioning

confidence: 99%

In vitro studies on the sexual maturation of the bovine lungworm Dictyocaulus viviparus during the development of preadult larvae to adult worms

2011

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The bovine lungworm Dictyocaulus viviparus is one of the most important parasites in grazing cattle. However, not much is known about morphology and molecular aspects of sexual maturation occurring during development of preadult larvae (L5) to adults. Since studies in the pulmonary compartments are infeasible, an in vitro cultivation method was established. The study was conducted with L5 during in vitro cultivation, assessing longitudinal growth and sexual maturation. Best results were achieved with RPMI-1640 medium with L-glutamine, 50% fetal bovine serum, amphotericin B (0.25 mg/ml), penicillin (10,000 U/ml), and streptomycin (10 mg/ml) at 39°C and 5% atmospheric CO₂. During cultivation, individuals grew from an average length of 4.64 to 9.88 mm independent of their density per setup. Regarding sexual maturation, female individuals started to lay eggs, whereas the testes of male individuals were filled with spermatozoa. Consequently, adult female and adult male worms developed. However, no copulation was observable and eggs did not embryonate. Development was further investigated by quantitative real-time PCR transcriptional analysis of major sperm protein (msp) and vitellogenin (vit) representing male and female sexual development, respectively. Male msp transcription peaked after 5 days of cultivation [corresponding to 20 days post infection (dpi)] and decreased gradually afterwards. Female vit transcription showed the highest rate after 15 days of cultivation (30 dpi), however it never reached the transcription rate in female adults isolated from the host. All in all, the present study gives not only insights into morphological differentiation but provides data lightening molecular aspects of sexual maturation in D. viviparus.

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“…Housekeeping genes, which are essential for the maintenance of the cell, are generally assumed to be stably expressed (10). However, a number of studies (8,(11)(12)(13) have shown that mRNA levels of housekeeping genes like beta-actin and GAPDH can vary in different conditions (14). Thus, housekeeping genes may not be appropriate standards for gene expression studies.…”

Section: Introductionmentioning

confidence: 99%

High expression stability of microtubule affinity regulating kinase 3 (MARK3) makes it a reliable reference gene

et al. 2010

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SummaryDifference in gene expressions is characteristic of the function of different cell types and those genes with low expression variance can be used as standards for quantitative gene expression studies. Microarray technology is used to study global gene expression within a cell; hence, represents a suitable source of data to mine for genes with low expression variance. The coefficient of variation (COV) of each gene was determined and a threshold of less than 0.1 COV was used to select stably expressed genes in each data set. Our results showed that microtubule affinity-regulating kinase 3 (MARK3) has the lowest COV in eight microarray datasets. In addition, the gene expression of housekeeping genes, which is very likely to be stably expressed, tends to fluctuate highly under different conditions, marking them as being less reliable for use as reference genes.

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Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus

Cited by 55 publications

References 21 publications

Molecular characterization and real-time PCR transcriptional analysis of Dictyocaulus viviparus major sperm proteins

Molecular characterization and real-time PCR transcriptional analysis of Dictyocaulus viviparus major sperm proteins

In vitro studies on the sexual maturation of the bovine lungworm Dictyocaulus viviparus during the development of preadult larvae to adult worms

High expression stability of microtubule affinity regulating kinase 3 (MARK3) makes it a reliable reference gene

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