Primer Express Software Version 3.0 Download

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Berry Spitsberg

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Jun 28, 2024, 8:11:07 PM6/28/24

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Quantitative real-time PCR (qPCR) is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA (gDNA). The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin (fALB). Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies.

Primer Express Software Version 3.0 Download

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To assess the cell number present in a PCR reaction, the coanalysis of suitable reference genes is crucial. Such reference genes should be single-copy number genes and should not frequently undergo genetic alterations, to allow the accurate normalization of genomic DNA (gDNA) samples. In addition, internal control genes are also used to investigate abnormalities in gene number, and amplified oncogenes have been shown to have diagnostic, prognostic, and therapeutic relevance. Thus, TaqMan PCR-based gene quantification assays are also used to identify allelic imbalances (germ-line deletions or amplifications), for example, in individuals suffering from breast cancer, cutaneous melanoma, or nervous system tumors [8, 9].

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) represents a universally expressed reference gene that has many biological roles in addition to its function in glycolysis. A GAPDH assay has been developed for the accurate normalization of feline messenger RNA (mRNA) expression [10], and this assay was recently validated and compared to other potential feline mRNA reference gene assays [11]. Subsequently, the GAPDH assay was also applied as quality control to test for the integrity of the gDNA and the absence of PCR inhibitors [10, 12, 13]. One report suggested that a single copy of the GAPDH pseudogene is present in the feline genome and that the feline GAPDH assay can therefore be used to quantify cell number in feline samples [14]. However, no information on the exact position or the sequence of this GAPDH pseudogene was provided [14]. In contrast, a variable number of GAPDH pseudogenes has been reported for other organisms [15, 16].

All cats included in this study were in experimental studies officially approved by the Veterinary Office of the Swiss Canton of Zurich (TVB 30/2003, 59/2005, and 99/2007). The cats were kept in groups under optimal ethological conditions in a barrier facility, as previously described [21]. All cats were euthanized for reasons unrelated to the present study.

The newly designed fALB qPCR assay was evaluated according to the MIQE guidelines [28] using the Rotor-Gene 6000 real-time rotary analyzer (Corbett). The efficiency of the assay was calculated as previously described [29], using the following equation: . The analytical sensitivity of the system was defined by an endpoint dilution experiment using the ten-fold serially diluted standard template and ten replicates per dilution, as described [27]. The linear range of amplification of the fALB qPCR assay was determined by ten-fold serial dilution of the linearized standard template. For the precision analysis, a dilution of a standard template containing 105 copies of fALB per reaction was chosen and assayed 10 and 13 times for the intrarun and interrun precision, respectively, and the mean value, standard deviation, and coefficients of variation were calculated.

An approach combining sequencing and bioinformatics was chosen to investigate the presence of potential GAPDH pseudogenes in the domestic cat genome. The potential target sequence of the GAPDH qPCR assay was amplified and cloned, and 15 clones were sequenced: 10 from cat B8 and 5 from cat 15. The sequencing of these 15 clones yielded 11 distinct sequences similar to that of the feline GAPDH mRNA (GenBank: NM_001009307). Eight different sequences were found within the target region of the primers and/or the hydrolysis probe of the GAPDH TaqMan assay (Figure 1). Overall, the sequence identity ranged from 82% (cat 15, clone 20: cat 15_20, Figure 1) to 96% (cat B8, clone 9: cat B8_9) in comparison to the reference sequence (GenBank: NM_001009307).

In this report, we describe the detection of several GAPDH-like sequences that are characteristic for processed GAPDH pseudogenes in the domestic cat genome. Based on the assumption that there is only one copy of the GAPDH pseudogene in the domestic cat genome [14], the GAPDH qPCR assay that was previously designed to amplify GAPDH mRNA/complementary DNA (cDNA) [10] was regularly used to determine the cell number of input gDNA. However, in our experience, the analysis of gDNA samples resulted in a lower amplification efficiency compared to cDNA (unpublished observations); we hypothesized that this might occur due to mismatches between the primers and/or hydrolysis probe and the gDNA sample. Thus, we performed a sequence analysis of the binding region of the primers and the hydrolysis probe of the GAPDH assay. The sequencing of 15 different clones comprising the GAPDH assay sequence revealed 11 different GAPDH-like sequences; however, none exhibited 100% similarity to the GAPDH mRNA sequence (GenBank: NM_001009307). It is possible that there are additional GAPDH pseudogenes present in the feline genome that were not detected in the present study because the sequencing was restricted to 15 clones. The use of an alternative sequencing technique (deep-sequencing rather than cloning followed by Sanger dideoxy sequencing) may provide a broader picture of the GAPDH-like sequences present in the cat genome. Furthermore, the primer binding sites chosen within the GAPDH sequence may have additionally restricted the number of recognized GAPDH pseudogenes. Thus, the number of GAPDH pseudogenes or GAPDH-like sequences in the feline genome may have been underestimated in our study. However, our sequencing results readily demonstrate that more than one feline GAPDH pseudogene is present in the genomic DNA of feline cells, and the GAPDH pseudogene sequences differed from the GAPDH mRNA sequence to some extent.

This study investigated the number of GAPDH pseudogenes in the domestic cat. Our results indicate that several closely related GAPDH-like sequences are indeed present in the cat genome. The GAPDH assay may still be used for quality control to test for the integrity of gDNA and the absence of PCR inhibitors. However, it appears to be a suboptimal choice for the quantification of gDNA equivalents. A newly designed assay using the fALB reference gene for the normalization of gDNA was validated and implemented. We recommend using this highly sensitive fALB qPCR assay for the normalization of input genomic DNA equivalents in future studies.

A. Katrin Helfer-Hungerbuehler performed the experiments, analyzed the data, and drafted the paper. Stefan Widmer performed the sequencing experiments as a partial fulfillment of the requirement of a term paper. RHL conceived and supervised the follow-up study and edited the paper.

The authors would particularly like to thank Dr. V. Cattori and Dr. M. L. Meli for excellent assistance and helpful discussions. They are grateful to the animal caretakers for expert technical aid with the cats and to the technicians and doctoral students of the Clinical Laboratory, particularly T. Meili Prodan and B. Weibel, and the Institute for Veterinary Pathology, Vetsuisse Faculty, for their excellent laboratory assistance. The molecular biology work was performed using the logistics of the Center for Clinical Studies, Vetsuisse Faculty, University of Zurich. This study was financially supported by a Research Grant from the Swiss National Science Foundation (310030_135586).

Linearity of the qPCR assay and absolute quantification of fALB amplification: The linearity of the qPCR assay was determined using a ten-fold serial dilution of the linearized standard template. In this Figure, representative results from a qPCR run using a Rotor-Gene 6000 real-time rotary analyzer (Corbett) are shown. A) Amplification plot of the fALB qPCR assay depicts cycle number versus normalized fluorescence. B) A standard curve (of a representative qPCR) shows the logarithmic starting input quantity (copies per reaction) of a 10-fold serial dilution of the standard template versus the measured cycle threshold (CT). The CT refers to the number of cycles required before the fluorescence passes a fixed threshold. Earlier increases in normalized fluorescence are associated with lower threshold cycle numbers and therefore higher starting quantities of sample template. The fALB assay showed linearity over 8 orders of magnitude. The correlation coefficient of the curve was 0.999, and the slope of the dilution versus threshold cycle curve was -3.33, which is ideal.

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This is the first in a set of articles giving an overview of the PCI Express (PCIe) protocol. This is quite a large subject and, I think, has the need to be split over a number of separate, more manageable documents and, even so, it is just a brief introduction to the subject. The main intended audience for these articles is anyone wishing to understand PCIe more if, say, they are working on a system which includes this protocol, or needs a primer before diving more deeply into the specifications themselves.