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Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, Ixodes scapularis (Acari: Ixodidae).

ABSTRACT: Obtaining reliable gene expression data using real-time quantitative polymerase chain reaction (qPCR) is highly dependent on the choice of normalization method. We tested the expression stability of multiple candidate genes in the salivary glands (SG) and synganglia (SYN) of female Ixodes scapularis (Say) ticks in multiple blood-feeding phases. We found that the amount of total RNA in both the SG and SYN increases dramatically during tick feeding, with 34x and 5.8x increases from 62 and 7.1 ng of unfed tick, respectively. We tested candidate genes that were predicted from I. scapularis genome data to encode glyceraldehyde 3-phosphate dehydrogenase (gapdh), ribosomal protein L13A (l13a), TATA box-binding protein (tbp), ribosomal protein S4 (rps4), glucose 6-phosphate dehydrogenase (gpdh), and beta-glucuronidase (gusb). The geNorm and NormFinder algorithms were used to analyze data from different feeding phases (i.e., daily samples from unfed to fully engorged females over a 7-d period in three replicate experiments). We found that the rps4 and l13a genes showed highly stable expression patterns over the feeding duration in both the SG and SYN. Furthermore, the highly expressed rps4 gene makes it useful as a normalization factor when we perform studies using minute amounts of dissected tissue for qPCR. We conclude that rps4 and l13a, whether individually or as a pair, serve as suitable internal reference genes for qRT-PCR studies in the SG and SYN of I. scapularis.

SUBMITTER: Koci J

PROVIDER: S-EPMC3703510 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, Ixodes scapularis (Acari: Ixodidae).

Koci Juraj J Simo Ladislav L Park Yoonseong Y

Journal of medical entomology 20130101 1

Obtaining reliable gene expression data using real-time quantitative polymerase chain reaction (qPCR) is highly dependent on the choice of normalization method. We tested the expression stability of multiple candidate genes in the salivary glands (SG) and synganglia (SYN) of female Ixodes scapularis (Say) ticks in multiple blood-feeding phases. We found that the amount of total RNA in both the SG and SYN increases dramatically during tick feeding, with 34x and 5.8x increases from 62 and 7.1 ng o ...[more]

PMID: 23427655

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Project description:Illumina and 454 pyrosequencing were used to characterize genes from the synganglion of female Ixodes scapularis. GO term searching success for biological processes was similar for samples sequenced by both methods. However, for molecular processes, it was more successful for the Illumina samples than for 454 samples. Functional assignments of transcripts predicting neuropeptides, neuropeptide receptors, neurotransmitter receptors and other genes of interest was done, supported by strong e-values (<-6), and high consensus sequence alignments. Transcripts predicting 15 putative neuropeptide prepropeptides ((allatostatin, allatotropin, bursicon α, corticotropin releasing factor (CRF), CRF-binding protein, eclosion hormone, FMRFamide, glycoprotein A, insulin-like peptide, ion transport peptide, myoinhibitory peptide, inotocin ( = neurophysin-oxytocin), Neuropeptide F, sulfakinin and SIFamide)) and transcripts predicting receptors for 14 neuropeptides (allatostatin, calcitonin, cardioacceleratory peptide, corazonin, CRF, eclosion hormone, gonadotropin-releasing hormone/AKH-like, insulin-like peptide, neuropeptide F, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin) are reported. Similar to Dermacentor variabilis, we found transcripts matching pro-protein convertase, essential for converting neuropeptide hormones to their mature form. Additionally, transcripts predicting 6 neurotransmitter/neuromodulator receptors (acetylcholine, GABA, dopamine, glutamate, octopamine and serotonin) and 3 neurotransmitter transporters (GABA transporter, noradrenalin-norepinephrine transporter and Na+-neurotransmitter/symporter) are described. Further, we found transcripts predicting genes for pheromone odorant receptor, gustatory receptor, novel GPCR messages, ecdysone nuclear receptor, JH esterase binding protein, steroidogenic activating protein, chitin synthase, chitinase, and other genes of interest. Also found were transcripts predicting genes for spermatogenesis-associated protein, major sperm protein, spermidine oxidase and spermidine synthase, genes not normally expressed in the female CNS of other invertebrates. The diversity of messages predicting important genes identified in this study offers a valuable resource useful for understanding how the tick synganglion regulates important physiological functions.

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Dataset Information

Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, Ixodes scapularis (Acari: Ixodidae).

Publications

Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, Ixodes scapularis (Acari: Ixodidae).

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