Abstract
Background
Comparative DNA microarray analyses typically yield very large gene expression data sets that reflect complex patterns of change. Despite the wealth of information that is obtained, the identification of stable reference genes is required for normalization of disease- or drug-induced changes across tested groups. This is a prerequisite in quantitative real-time reverse transcription-PCR (qRT-PCR) and relative RT-PCR but rare in gene microarray analysis. The goal of the present study was to outline a simple method for identification of reliable reference genes derived from DNA microarray data sets by comparative statistical analysis of software-generated and manually calculated candidate genes.
Material/Methods
DNA microarray data sets derived from whole-blood samples obtained from 14 Zucker diabetic fatty (ZDF) rats (7 lean and 7 diabetic obese) were used for the method development. This involved the use of software-generated filtering parameters to accomplish the desired signal-to-noise ratios, 75th percentile signal manual normalizations, and the selection of reference genes as endogenous controls for target gene expression normalization.
Results
The combination of software-generated and manual normalization methods yielded a group of 5 stably expressed, suitable endogenous control genes which can be used in further target gene expression determinations in whole blood of ZDF rats.
Conclusions
This method can be used to correct for potentially false results and aid in the selection of suitable endogenous control genes. It is especially useful when aimed to aid the software in cases of borderline results, where the expression and/or the fold change values are just beyond the pre-established set of acceptable parameters.
MeSH Keywords: Data Interpretation, Statistical; Diabetes Mellitus, Type 2; Microarray Analysis
Background
The generation of very large amounts of gene microarray data poses a challenge, not only for its processing but also for its interpretation, due to intrinsic false discovery rates. In addition, the problem of background noise along with differences in hybridization efficiencies is also an important factor generating variability within and among microarray chips, constituting major confounding elements in gene expression analysis. Some of the most advanced commercially available software can automatically account for most, but not all, of these challenges.
In general, the persistence of confounding elements generates the need for appropriate data normalization methods, such as using the specific nth percentile signal intensity value of a particular array. Often, software-generated normalization methods have already incorporated the nth-percentile approach (e.g., 75th percentile) along with some background-subtraction mechanism [1]. In this regard, dealing with the assay’s inherent background noise becomes critical to account for signal stringency. Hence, the importance of using filtering parameters to accomplish the desired signal-to-noise ratios becomes obvious.
Moreover, it becomes necessary to use housekeeping or reference genes as endogenous controls for further gene signal normalization. This is not a common use in gene microarray analysis, where log transformation, background subtraction, and nth percentile normalizations have been the norm [1]. In this regard, the use of endogenous control genes is prerequisite in qRT-PCR and relative RT-PCR [2–4]. The principle behind this methodology consists of simply using widely expressed genes that do not respond to most treatments as references to compare to genes of interest (target genes) that do change. This helps in the proper interpretation of gene expression patterns and in calculating relative gene expression fold changes between treatment groups, minimizing technique-derived experimental errors. Thus, the same rationale should apply in gene microarray analysis. However, selecting the right endogenous control genes for normalizing data can be difficult since these widely expressed reference genes are not truly universal. In this regard, there have been observed changes in reference gene expression with different treatments as well as tissue-specific differential reference gene expression patterns [2–10]. For this reason, a systematic method must be determined for its use in the selection of array-specific (i.e., tissue- and taxa-specific) endogenous control genes based on a pool or pools of pre-established and widely used housekeeping or reference genes.
In the present examination, dependent measure data sets were derived from paired DNA microarray gene expression analyses performed on whole-blood samples from homozygous ZDF rats exhibiting clinically-relevant type 2 symptomatology in comparison to heterozygous healthy lean controls [11]. In this regard, the ZDF rat has been well established in the biomedical literature as a high-resolution translational model for elucidation of underlying pathophysiological mechanisms critically linked to advanced therapeutic development for major human disorders, including type 2 diabetes [12,13], cardiovascular disease [14], renal disease [15], atherosclerosis [16–18], and rheumatoid arthritis [11]. In addition, a list of potentially suitable endogenous control genes for the study of whole-blood ZDF rat samples is provided.
Material and Methods
The analytical software used in this examination was Agilent’s GeneSpring GX, ver.13.1.1. Manual calculations were performed using the Microsoft Excel basic package. The gene microarray data (Agilent single-color expression) was obtained from a published study [11] using whole-blood samples collected from 7 twelve-week-old male homozygous (Fa/Fa) leptin receptor-deficient ZDF rats exhibiting a full-fledged type 2 diabetic phenotype highlighted by hyperglycemia, hyperlipidemia, liver hypertrophy, increased water consumption, and urine output, and from 7 twelve-week-old male heterozygous (Fa/fa) healthy lean controls. Briefly, the study animals were housed 2 per cage and maintained in an Innovive caging system (San Diego, CA). The rooms were lit for 12 hours from 7:00 AM to 7:00 PM, each day, using artificial light. Animals had free access to water and Purina 5008 rodent food (Waldschimdt’s, Madison, WI) for the duration of the study (7 weeks) [11]. The study was approved by the Institutional Animal Care and Use Committee (IUCAC, Study Number SNY1301). Animal care and all technical procedures were performed by PhysioGenix, Inc. staff in accordance with the established protocols in the National Institute of Health Guide for Care and Use of Laboratory Animals (Eighth Edition).
The initial data were processed using Agilent’s feature extraction software, followed by analysis using the microarray platform software GeneSpring and by enhancement through a manual optimization method, as follows:
First, using the microarray software, the following filtering parameters were implemented: a filter by flags (e.g., “detected”, “not detected”, and “compromised”) where irregular features (or signals) were discarded, and a signal-to-noise ratio of 2, which was chosen as the lower limit in at least 1 of the subject groups. In addition, a list of 34 annotated reference genes previously identified in the biochemical literature [2–8,19] was built and used to filter the experimental data.
Second, the signal intensities of the reference genes passing the above-mentioned filters were manually divided by the 75th percentile value of the corresponding arrays and the resulting values were used to calculate fold changes in gene expression between the healthy lean and the diabetic obese groups by simple division (i.e., diabetic obese value/healthy lean value). It should be noted that a gene variation was deemed biologically irrelevant when its fold change value was defined as −1.2<x<1.2.
Statistical analyses
Software-generated data was compared using moderated t test method with Benjamini-Hochberg multiple testing correction. The t test was used for evaluating the manually normalized data.
Results
Application of filtering parameters
The first step before data analysis deals with signal quality control and the setting of filtering parameters in a particular data set. In this evaluation, the filters previously described were used to identify potential endogenous control gene candidates from the list of 34 widely used reference genes [2–8,19] (Table 1). After this initial filtering process, in which genes not meeting the signal quality criteria were filtered out (i.e., flagged as “compromised”; S/N <2), a working list of 18 gene probes corresponding to 16 endogenous gene candidates was made (Table 2). It should be noted that there can be more than 1 probe per gene, each having a different sequence, thus hybridizing to a different region of the gene transcript.
Table 1.
List of commonly used reference genes.
| Gene symbol | Gene name | Chromosome |
|---|---|---|
| A4galt | alpha 1,4-galactosyltransferase | chr7 |
| Actb | Actin, beta | chr12 |
| B2m | beta-2 microglobulin | chr3 |
| Cck | Cholecystokinin | chr8 |
| Cry2 | Cryptochrome 2 (photolyase-like) | chr3 |
| Csnk1g2 | Casein kinase 1, gamma 2 | chr7 |
| Decr1 | 2,4-dienoyl CoA reductase 1, mitochondrial | chr5 |
| Dimt1 | DIM1 dimethyladenosine transferase 1 homolog (S. cerevisiae) | chr2 |
| Eef1a1 | Eukaryotic translation elongation factor 1 alpha 1 | chr8 |
| Farp1 | FERM, RhoGEF (Arhgef) and pleckstrin domain protein 1 (chondrocyte-derived) | chr15 |
| Fpgs | Folylpolyglutamate synthase | chr3 |
| Gapdh | Glyceraldehyde-3-phosphate dehydrogenase | chr4 |
| Gins2 | GINS complex subunit 2 (Psf2 homolog) | chr19 |
| Gusb | Glucuronidase, beta | chr12 |
| Hmbs | Hydroxymethylbilane synthase | chr8 |
| Hprt1 | Hypoxanthine phosphoribosyltransferase 1 | chrX |
| Hsp90ab1 | Heat shock protein 90 alpha (cytosolic), class B member 1 | chr9 |
| Mapre2 | Microtubule-associated protein, RP/EB family, member 2 | chr18 |
| Pex16 | Peroxisomal biogenesis factor 16 | chr3 |
| Pgk1 | Phosphoglycerate kinase 1 | chrX |
| Polr2a | Polymerase (RNA) II (DNA directed) polypeptide A | chr10 |
| Ppia | Peptidylprolyl isomerase A (cyclophilin A) | chr14 |
| Ppib | Peptidylprolyl isomerase B | chr8 |
| Pum1 | Pumilio RNA-binding family member 1 | chr5 |
| Rpl4 | Ribosomal protein L4 | chr8 |
| Rplp2 | Ribosomal protein, large P2 | chr1 |
| Sdha | Succinate dehydrogenase complex, subunit A, flavoprotein (Fp) | chr1 |
| Srsf4 | Serine/arginine-rich splicing factor 4 | chr5 |
| Tbp | TATA box binding protein | chr1 |
| Tfrc | Transferrin receptor | chr11 |
| Trap1 | TNF receptor-associated protein 1 | chr10 |
| Ubc | Ubiquitin C | chr12 |
| Ywhag | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma | chr12 |
| Ywhaz | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta | chr7 |
List of 34 widely used housekeeping/reference genes screened for endogenous control gene selection.
Table 2.
Filtered reference gene candidates for endogenous control gene selection.
| Probe name | Gene symbol | Gene name | Fold change | p-value | Chromosome |
|---|---|---|---|---|---|
| A_64_P050964 | Cry2 | Cryptochrome 2 (photolyase-like) | −1.46 | 7.40×10−3 | chr3 |
| A_42_P526030 | Decr1 | 2,4-dienoyl CoA reductase 1, mitochondrial | −1.32 | 1.91×10−2 | chr5 |
| A_44_P524471 | Dimt1 | DIM1 dimethyladenosine transferase 1 homolog (S. cerevisiae) | −1.26 | 1.60×10−1 | chr2 |
| A_64_P232432 | Gapdh | Glyceraldehyde-3-phosphate dehydrogenase | −2.83 | 2.20×10−3 | chr4 |
| A_64_P052510 | Gapdh | Glyceraldehyde-3-phosphate dehydrogenase | −2.88 | 3.40×10−3 | chr4 |
| A_64_P073003 | Gusb | Glucuronidase, beta | −1.34 | 1.90×10−3 | chr12 |
| A_44_P421363 | Hmbs | Hydroxymethylbilane synthase | −2.38 | 3.00×10−4 | chr8 |
| A_43_P11257 | Hprt1 | Hypoxanthine phosphoribosyltransferase 1 | −2.30 | 7.00×10−4 | chrX |
| A_64_P045716 | Hsp90ab1 | Heat shock protein 90 alpha (cytosolic), class B member 1 | 1.08 | 1.99×10−2 | chr9 |
| A_64_P140020 | Hsp90ab1 | Heat shock protein 90 alpha (cytosolic), class B member 1 | −1.60 | 8.30×10−3 | chr9 |
| A_64_P047724 | Mapre2 | Microtubule-associated protein, RP/EB family, member 2 | −1.85 | 3.50×10−2 | chr18 |
| A_42_P492082 | Pex16 | Peroxisomal biogenesis factor 16 | 2.31 | 7.40×10−3 | chr3 |
| A_64_P058353 | Ppia | Peptidylprolyl isomerase A (cyclophilin A) | −1.80 | 5.30×10−3 | chr14 |
| A_43_P13976 | Ppib | Peptidylprolyl isomerase B | −1.71 | 3.00×10−4 | chr8 |
| A_42_P767897 | Pum1 | Pumilio RNA-binding family member 1 | −1.11 | 1.28×10−2 | chr5 |
| A_64_P080678 | Sdha | Succinate dehydrogenase complex, subunit A, flavoprotein (Fp) | −1.67 | 3.60×10−3 | chr1 |
| A_42_P816010 | Srsf4 | Serine/arginine-rich splicing factor 4 | −1.08 | 3.96×10−1 | chr5 |
| A_44_P416641 | Ywhaz | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta | −2.71 | 9.00×10−4 | chr7 |
List of 18 gene probes resulting from software-generated filtering of 34 widely used housekeeping/reference genes. The fold change and p-values represent the variation between diabetic obese and healthy lean subjects. Statistics: Moderated t-test method with Benjamini-Hochberg Multiple Testing Correction.
Endogenous control gene determination
Software-generated gene selection
Suitable endogenous control genes should exhibit minimal-to-no expression variation between groups (e.g., control vs. treatment), in this particular study, between diabetic obese and healthy lean groups. In this evaluation, an absolute fold change value of 1.2 was set as the limit for the gene selection criterion. In this way, further filtering by fold change yielded 3 suitable endogenous control gene candidates, which can be seen in Figure 1. In addition, Table 3 shows these software-selected genes (Hsp90ab1, Pum1, and Srsf4) along with their fold change and p-values.
Figure 1.
Scatter Plot Visualization of Software-generated Endogenous Control Gene Candidates. Scatter plot comparison between healthy (Lean) and diabetic obese (ZDF) rats. Outer lines delimit fold change values ≤1.2 and ≥−1.2. Highlighted in blue are those genes with the least modulation differences between the two groups.
Table 3.
Software-generated endogenous control gene candidates.
| Probe name | Gene symbol | Gene name | Fold change | p-value | Chromosome |
|---|---|---|---|---|---|
| A_64_P045716 | Hsp90ab1 | Heat shock protein 90 alpha (cytosolic), class B member 1 | 1.08 | 1.99×10−2 | chr9 |
| A_42_P816010 | Srsf4 | Serine/arginine-rich splicing factor 4 | −1.08 | 3.96×10−1 | chr5 |
| A_42_P767897 | Pum1 | Pumilio RNA-binding family member 1 | −1.11 | 1.28×10−2 | chr5 |
The use of Agilent’s GeneSpring GX 13.1.1 software yielded 3 housekeeping/reference genes as suitable candidates for endogenous control genes. The fold change and p-values represent the variation between diabetic obese and healthy lean subjects. Statistics: Moderated t-test method with Benjamini-Hochberg multiple testing correction.
Manual normalization
The manual method involving 75th percentile normalization of background-subtracted signals along with fold change calculations yielded 5 potentially suitable endogenous control candidates. Three were the same as the software-generated genes, plus 2 additional genes – Dimt1 and Gusb (Table 4). These genes exhibited fold change values <1.2 and >−1.2, with p-values considered not significant (p>0.05). In this regard, after manual normalization, there was an additional gene, Decr1, that exhibited an acceptable fold change value of 1.19 but had a p-value <0.05 and hence was not selected (Table 2). Table 5 shows an arbitrary gene grouping based on signal intensity values (low, medium, high). This helps in the selection of suitable endogenous control genes because, as mentioned earlier, these genes should ideally be chosen so that they encompass a large signal intensity spectrum in such a way that it compensates for the potentially diverse copy numbers of target genes (translated as signal intensities) [3]. Finally, Table 6 shows the Agilent probe sequences of each of the endogenous control genes selected by this method.
Table 4.
Endogenous control genes selected after manual normalization.
| Probe name | Gene symbol | Gene name | Software generated | Manually normalized | Chromosome | ||
|---|---|---|---|---|---|---|---|
| Fold change | p-value | Fold change | p-value | ||||
| A_44_P524471 | Dimt1 | DIM1 dimethyladenosine transferase 1 homolog (S. cerevisiae) | −1.26 | 1.60×10−1 | −1.13 | 3.46×10−1 | chr2 |
| A_64_P073003 | Gusb | Glucuronidase, beta | −1.34 | 1.90×10−3 | −1.19 | 9.82×10−2 | chr12 |
| A_64_P045716 | Hsp90ab1 | Heat shock protein 90 alpha (cytosolic), class B member 1 | 1.08 | 1.99×10−2 | 1.16 | 2.19×10−1 | chr9 |
| A_42_P767897 | Pum1 | Pumilio RNA-binding family member 1 | −1.11 | 1.28×10−2 | −1.01 | 9.58×10−1 | chr5 |
| A_42_P816010 | Srsf4 | Serine/arginine-rich splicing factor 4 | −1.08 | 3.96×10−1 | 1.02 | 9.28×10−1 | chr5 |
Manual normalization yielded 2 additional genes, Dimt1 and Gusb, to the original list of 3 software-generated endogenous control candidates. Statistics: Student t-test was used for evaluating the manually normalized data.
Table 5.
Gene expression levels by signal intensity value.
| Probe name | Gene symbol | Fold change | p-value | Manually normalized signal | Chromosome |
|---|---|---|---|---|---|
| A_64_P232432 | Gapdh | −2.42 | 3.35×10−4 | 14.85 | chr4 |
| A_64_P052510 | Gapdh | −2.46 | 9.48×10−4 | 14.48 | chr4 |
| A_64_P047724 | Mapre2 | −1.61 | 1.34×10−2 | 11.41 | chr18 |
| A_64_P058353 | Ppia | −1.65 | 6.42×10−3 | 6.53 | chr14 |
| A_44_P416641 | Ywhaz | −2.38 | 4.88×10−5 | 6.29 | chr7 |
| A_43_P13976 | Ppib | −1.53 | 5.73×10−4 | 6.11 | chr8 |
| A_64_P045716 | Hsp90ab1 | 1.16 | 2.19×10−1 | 6.07 | chr9 |
| A_44_P421363 | Hmbs | −1.99 | 7.29×10−3 | 4.18 | chr8 |
| A_43_P11257 | Hprt1 | −2.08 | 9.15×10−5 | 3.44 | chrX |
| A_42_P816010 | Srsf4 | 1.02 | 9.28×10−1 | 2.65 | q |
| A_64_P140020 | Hsp90ab1 | −1.44 | 5.42×10−4 | 2.58 | chr9 |
| A_64_P080678 | Sdha | −1.49 | 4.16×10−4 | 2.42 | chr1 |
| A_42_P526030 | Decr1 | −1.19 | 2.77×10−2 | 1.26 | chr5 |
| A_42_P492082 | Pex16 | 3.49 | 1.24×10−1 | 0.4 | chr3 |
| A_64_P073003 | Gusb | −1.19 | 9.82×10−2 | 0.33 | chr12 |
| A_64_P050964 | Cry2 | −1.27 | 6.54×10−2 | 0.28 | chr3 |
| A_44_P524471 | Dimt1 | −1.13 | 3.46×10−1 | 0.27 | chr2 |
| A_42_P767897 | Pum1 | −1.01 | 9.58×10−1 | 0.22 | chr5 |
Selected endogenous control candidate genes shown in bold font. Signal Intensity: 10–15 = high
; 1–9.99 = med
; 0–0.99 = low
. Arbitrary signal intensity-based separation of mean background-subtracted signals normalized by their corresponding microarray’s 75th-percentile value. Note that the genes selected (in bold) show fold changes between 1.2 and −1.2, with p-values greater than 0.05, indicating that changes in expression were not statistically significant. Also note that although gene Decr1 exhibits a fold change below 1.2, its p-value is lower than 0.05 and therefore this gene was not selected. Statistics: Student t-test was used for evaluating the manually normalized data.
Table 6.
Gene probe sequences.
| Probe name | Gene symbol | Sequence |
|---|---|---|
| A_44_P524471 | Dimt1 | CAGAAGATTTCAGTATAGCCGATAAAATACAGCAGATCCTAACCAACACAGGTTTTAGTG |
| A_64_P073003 | Gusb | AGAGGTTACGGTTCAGTGCCGAGGACCCAGTGTATGGGAAGCAGACCGTTCACATTCTAA |
| A_64_P045716 | Hsp90ab1 | TCTCATGAAGGAGACACAGAAGTCCATCTACTATATCACTGGTGAGAGCAAAGAGCAGGT |
| A_42_P767897 | Pum1 | AAGTACACCTATGGCAAGCACATCCTGGCCAAGCTTGAGAAGTACTACATGAAGAATGGT |
| A_42_P816010 | Srsf4 | CTTGTGAATAGCACAGTCAAGAGAAATGGATACCTGCATAGCCCATAGGAAGTAACACTG |
This table shows the gene probe sequences for Rattus norvegicus, corresponding to Agilent’s microarray technology.
Discussion
Filtering parameters to accomplish the desired signal-to-noise ratios
Gene microarray data need to be adequately filtered. The first step before data analysis involved a quality control step in which irregular signals or “compromised” features are removed (i.e., filter by flags: detected; not detected; compromised). Often, in order to accomplish an acceptable microarray signal intensity level, a signal should be at least twice as strong as that of the background (i.e., signal-to-noise ratio ≥2) and, depending on the desired stringency level, this filter cut-off can be set to a signal-to-noise ratio of 3 or higher. Normally, gene microarray technologies produce a consistent background signal whose mean level information can be easily obtained from the raw image data (e.g., using feature extraction software). A microarray platform’s software automatically subtracts calculated background signal from raw signal values, effectively yielding processed raw signal values. To filter out genes whose processed raw signal values are less than twice the background (S/N <2), a filter (i.e., processed raw signal cut-off) should be set to a lower limit equivalent to the array’s mean background signal value. In this way, if the processed (i.e., background-subtracted) raw signal is added to the technology’s mean background signal value, it will be equivalent to S/N=2. Processed data falling below the S/N=2 level were eliminated from the final data set. Moreover, this filtering by expression level was applied so it would accept a gene when in at least 1 of the subject groups studied (e.g., control; treatment “A”; and treatment “B”) is detectable since, for example, a given treatment/s could cause downregulation of a gene below a level corresponding to S/N=2. The same applies when a gene is only detectable after a treatment. In this way, when a particular gene or group of genes was present at S/N ≥2 in at least 1 of the subject groups, then those genes passed the filter.
Endogenous control gene selection and normalization
As mentioned earlier, selecting the right endogenous control genes for data normalization can be difficult due to gene expression changing with treatments or to tissue-specific differential gene expression patterns [4,9,10]. For this purpose, based on several important publicly available studies [2–8,19], a list of 34 widely used reference genes was built to be evaluated with the experimental data.
Ideally, it is preferable to select more than 1 endogenous control gene and to average their values. In this regard, it is recommended that, when possible, the endogenous control genes be chosen so that they will exhibit different signal intensity levels (e.g., low, medium, and high) [3]. This would account for the differences in copy number (i.e., signal intensities) among the target genes. Hence, the signal intensity levels of the potential endogenous control gene candidates were compared to be sure they spanned a relatively wide range. Moreover, suitable endogenous control genes should be selected such that each is involved in a different cellular function and/or is found in different chromosomes [5,6]. Although this may not always be possible, it is recommended that at least 2 of these criteria be satisfied (Table 5).
Finally, in order to overcome or minimize inter-array differences, scaling to the nth percentile is recommended (in this case, to the 75th percentile) [1]. If using a linear scale (i.e., not log-normalized), as in this case, the processed (background-subtracted) signal intensity values are divided by the 75th percentile value corresponding to the particular array. This scaling is applied to both potentially suitable endogenous control genes and target genes.
Normalization of target genes
After finding and normalizing suitable endogenous control genes, the next step is to use them to normalize genes of interest to calculate their expression pattern though fold change values. In this regard, the problem with software-generated microarray gene signal intensities becomes more evident at the time of their fold change determination. This challenge is not only observed with the calculated fold changes, but also with the corresponding p-values, which may not be statistically significant (e.g., >0.05) and hence, relevant genes may be filtered out. However, with the normalization method described above, along with the utilization of reliable endogenous control genes, this can be corrected.
One important step taken before the analysis of gene expression is to restrict the search to a specific gene list or lists pertaining to a more focused field of interest (e.g., a particular disease-related list of genes). This helps in the manageability of the data set by restricting it to a much lower number of genes. The next step will be to filter the data according to the filtering parameters depicted in the previous section. That is, filtering by flags, leaving out those having compromised signals, and then filtering by expression, leaving out genes whose processed (background-subtracted) raw signal values are less than twice the background (S/N <2). Again, this is achieved by setting the processed signal’s lower limit to the equivalent of the technology’s mean background signal value. Once this selected group of genes is filtered, the next step is to manually scale the gene’s processed raw signals to the 75th percentile, as described above. The resulting target gene values are then normalized by simple division using the combined value (i.e., mean) of the endogenous control or reference genes selected earlier, as follows: target gene value/endogenous control mean value, for each target gene. In this way, the values obtained can be used to compare control and treatment groups through fold change calculations (e.g., treated vs. control group) and the calculated p-values used to evaluate their statistical significance.
Conclusions
The ZDF rat is a proven model for the study of different comorbidities associated with type 2 diabetes. The results obtained in the present study demonstrate how use of a simple combination of software-generated and manual normalization methods can correct for potentially false results and aid in the selection of suitable endogenous control genes to be used in further gene expression determinations; in the present case, in the study of ZDF rat whole-blood samples. The expression of these genes showed no statistically significant differences between homozygous ZDF rats exhibiting clinically-relevant type 2 symptomatology and the heterozygous healthy lean controls, a characteristic which rendered them suitable. Importantly, the endogenous control genes that were found constitute a reliable platform for use in gene expression studies aiming to evaluate potentially novel therapeutic interventions for treatment of comorbidities and their progression in human populations with type 2 diabetes.
This method is especially useful when aimed to aid the software in cases of borderline results, where the expression and/or the fold change values are just beyond the pre-established set of acceptable parameters. In this regard, the difference between a gene with a p-value of 0.049 and one with a p-value of 0.051 is meaningless per se, as their true relevance is their biological significance. Hence, the use of endogenous control genes for the normalization of target genes assists in accomplishing the identification of potential biological significance in gene expression patterns. Moreover, this method should be applied every time in every array studied since, as noted earlier, differences in hybridization efficiencies along with changes in gene expression with different treatments and tissue-specific differential gene expression patterns are common occurrences.
Footnotes
Source of support: Departmental sources
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