Stable Internal Control Genes Research Articles

Identification of suitable internal control genes for gene expression analysis of banana in response to BBTV infection

Banana is one of the most abundant crops produced annually in the Philippines. The presence of banana bunchy top virus (BBTV) leading to banana bunchy top disease is one of the factors hindering the continuous production of the fruit crop. The use of an appropriate and stable internal control gene as reference in validation of differentially-expressed genes in an organism is important. This study aims to identify appropriate internal control genes for differential gene expression analysis in Musa balbisiana and Musa acuminata specific for BBTV infection. RNA extraction, complementary DNA (cDNA) synthesis and RT-qPCR (quantitative real time polymerase chain reaction) of BBTV-resistant and BBTV-susceptible Musa genotypes were performed. The RT-qPCR quantification data were then subjected to analysis on RefFinder software and geomean ranking values were calculated along with the four statistical algorithms (delta Cq, Genorm, BestKeeper and NormFinder). Based on the comprehensive ranking values in the software, L2 gene was the most suitable internal control gene for the differential expression analysis of both BBTV-resistant and BBTV-susceptible banana accessions. The internal control gene is recommended for the validation of selected candidate resistance and host factor genes in response to BBTV infection.

Evaluation of Reference Genes for Quantitative PCR in Eustoma grandiflorum under Different Experimental Conditions

Eustoma grandiflorum, commonly known as prairie gentian or Texas bluebells, is among the most popular agriculturally propagated species of cut flowers. Due to its widespread appeal, there is increasing interest in understanding the molecular genetic factors underlying floral development and resistance to abiotic stresses. We analyzed 18 potential reference genes in different organs, at different floral developmental stages and under drought- and salt-stress treatments, for use in RT-qPCR analysis. A total of four analytical tool packages, including geNorm, NormFinder, BestKeeper, and RefFinder were employed to determine the most appropriate reference genes under each treatment condition. The results demonstrate that different reference genes should be used for normalization under different experimental treatments. EgPP and EgPP2A2 were the most stable internal control genes across different organ types, EgPP and Eg18S were the most stable under salt-stress, EgPP and EgACT1 were the most stable across different floral development stages, and EgEF1A and EgTUA were the most stable reference genes under drought-stress. Additional gene expression analyses of EgMIXTA1, EgTOE1, and EgP5CS1 further confirmed the applicability of these reference genes. The results represent a significant contribution to future studies of reference gene selection for the normalization of gene expression in Eustoma grandiflorum.

Validation of superior reference genes for qRT-PCR and Western blot analyses in marine Emiliania huxleyi-virus model system.

To search for a set of reference genes for reliable gene expression analysis in the globally important marine coccolithophore Emiliania huxleyi-virus model system. Fifteen housekeeping genes (CDKA, CYP15, EFG3, POLAI, RPL30, RPL13, SAMS, COX1, GPB1-2, HSP90, TUA, TUB, UBA1, CAM3 and GAPDH) were evaluated for their stability as potential reference genes for qRT-PCR using ΔCt, geNorm, NormFinder, Bestkeeper and RefFinder software. CDKA, TUA and TUB genes were tested as loading controls for Western blot in the same sample panel. Additionally, target genes associated with cell apoptosis, that is metacaspase genes, were applied to validate the selection of reference genes. The analysis results demonstrated that putative housekeeping genes exhibited significant variations in both mRNA and protein content during virus infection. After a comprehensive analysis with all the algorithms, CDKA and GAPDH were recommended as the most stable reference genes for E huxleyi virus (EhV) infection treatments. For Western blot, significant variation was seen for TUA and TUB, whereas CDKA was stably expressed, consistent with the results of qRT-PCR. CDKA and GAPDH are the best choice for gene and protein expression analysis than the other candidate reference genes under EhV infection conditions. The stable internal control genes identified in this work will help to improve the accuracy and reliability of gene expression analysis and gain insight into complex E. huxleyi-EhV interaction regulatory networks.

Determination of the reference genes for qRT-PCR normalization and expression levels of MAT genes under various conditions in Ulocladium.

The genus Ulocladium is thought to be strictly asexual. One of the possible reasons for the lack of sexuality in Ulocladium species is the absence of the stimulus of environmental factors. Sexual reproduction in ascomycetes is controlled by a specific region in the genome referred to as mating-type locus (MAT) that consists of two dissimilar DNA sequences in the mating partners, termed MAT1-1 and MAT1-2 idiomorphs. To identify the response of MAT loci to environmental conditions, the mRNA transcription level of MAT1-1-1 and MAT1-2-1 genes was tested using qRT-PCR under different temperatures (−20 °C, −10 °C, 0 °C, 10 °C, 20 °C, 30 °C and 40 °C), culture medias (CM, OA, HAY, PCA, PDA and V8), photoperiods (24 h light, 24 h dark, 12 h light/12 h dark, 10 h light/14 h dark and 8 h light/16 h dark), and CO2 concentrations (0.03%, 0.5%, 1%, 5%, 10%, 15% and 20%). For obtaining reliable results from qRT-PCR, the most stable internal control gene and optimal number of reference genes for normalization were determined under different treatments. The results showed that there is no universal internal control gene that is expressed at a constant level under different experimental treatments. In comparison to various incubation conditions, the relative expression levels of both MAT genes were significantly increased when fungal mycelia were grown on HAY culture media at 0–10 °C with a light/dark cycle, indicating that temperature, culture media, and light might be the key environmental factors for regulating the sexuality in Ulocladium. Moreover, MAT1-1-1 and MAT1-2-1 genes showed similar expression patterns under different treatments, suggesting that the two MAT genes might play an equally important role in the sexual evolutionary process.

Identification of stable internal control genes for accurate normalization of real-time quantitative PCR data in testicular tissue from two breeds of cattle

Objective: To assess the stability of 10 candidate internal control genes (ICGs), namely GAPDH, ACTB, RPL23, RPS15A, ATPSF1, GLUT5, HMBS, ATP2B4, PPIA, and BRP to normalize the transcriptional data from testes samples of Zebu and crossbred bulls. Methods: Total RNA was isolated from testicular tissue of Zebu and crossbred bulls (n=6 each) between 2-8 years of age. cDNA was synthesized, and the quantitative real-time polymerase chain reaction (PCR) was performed. The cycle threshold values were used for the analysis of the stability of ICGs. Four different statistical algorithms: geNorm, Normfinder, BestKeeper, and RefFinder, were used to assess the stability of these genes. Results: ATPSF1, HMBS, PPIA, and RPS15A were the most reliable and stable ICGs for Zebu testes, and ATPSF1, RPL23, and PPIA for crossbred testes. Conclusions: A panel of stable ICGs (ATPSF1, HMBS, PPIA, RPS15A for Zebu and ATPSF1, RPL23, and PPIA for crossbred) for normalization of gene expression data in testes samples can be helpful for researchers to conduct functional genomics studies at the testicular level in cattle bulls.

Reference genes for accurate evaluation of expression levels in Trichophyton interdigitale grown under different carbon sources, pH levels and phosphate levels

Tinea pedis is a type of dermatophytosis caused by anthropophilic keratinolytic fungi such as Trichophyton interdigitale. Quantitative reverse transcription PCR (RT-qPCR) is a reliable and reproducible technique for measuring changes in target gene expression across various biological conditions. A crucial aspect of accurate normalization is the choice of appropriate internal controls. To identify reference genes for accurate evaluation of expression levels in T. interdigitale, the transcription levels of eight candidate reference genes (adp-rf, β-act, ef1-α, gapdh, psm1, sdha, rpl2 and ubc) and one target gene (Tri m4) were analysed by RT-qPCR after growing the dermatophyte under different environmental conditions. The results obtained from expression stability evaluations with NormFinder, geNorm, BestKeeper, and RefFinder software demonstrated that adp-rf and psm1 were the most stable internal control genes across all experimental conditions. The present study constitutes the first report of the identification and validation of reference genes for RT-qPCR normalization for T. interdigitale grown under different environmental conditions resembling the conditions encountered by fungi during invasion of skin.

Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica fortune

BackgroundIsatis indigotica, a traditional Chinese medicine, produces a variety of active ingredients. However, little is known about the key genes and corresponding expression profiling involved in the biosynthesis pathways of these ingredients. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful, commonly-used method for gene expression analysis, but the accuracy of the quantitative data produced depends on the appropriate selection of reference genes.ResultsIn this study, the systematic analysis of the reference genes was performed for quantitative real-Time PCR normalization in I. indigotica. We selected nine candidate reference genes, including six traditional housekeeping genes (ACT, α-TUB, β-TUB, UBC, CYP, and EF1-α), and three newly stable internal control genes (MUB, TIP41, and RPL) from a transcriptome dataset of I. indigotica, and evaluated their expression stabilities in different tissues (root, stem, leaf, and petiole) and leaves exposed to three abiotic treatments (low-nitrogen, ABA, and MeJA) using geNorm, NormFinder, BestKeeper, and comprehensive RefFind algorithms. The results demonstrated that MUB and EF1-α were the two most stable reference genes for all samples. TIP41 as the optimal reference gene for low-nitrogen stress and MeJA treatment, while ACT had the highest ranking for ABA treatment and CYP was the most suitable for different tissues.ConclusionsThe results revealed that the selection and validation of appropriate reference genes for normalizing data is mandatory to acquire accurate quantification results. The necessity of specific internal control for specific conditions was also emphasized. Furthermore, this work will provide valuable information to enhance further research in gene function and molecular biology on I. indigotica and other related species.

β-actin gene expression is variable among individuals and not suitable for normalizing mRNA levels in Portunus trituberculatus

The housekeeping gene encoding β-actin appears to be the most widely-used internal reference for gene expression studies in experimental animals or their cell lines. However, the effectiveness of β-actin to normalize mRNA levels expression in many crustacean species is still object of debate. To date, it is still unclear if β-actin is suitable to be utilized as the internal reference in qualitative real-time gene expression study in crab species. To address this concern, we evaluated 5 candidate reference genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A, elongation factor 1-α (EF1-α), and 18 S ribosomal RNA (18 S rRNA) in the swimming crabs (Portunus trituberculatus) models. Our data showed that the β-actin gene expression varied significantly across individual swimming crab individuals in gills or hemocytes and the expression of 18 S rRNA, EF1-α, cyclophilin or GAPDH gene were relatively stable compared to that of β-actin. Moreover, the expression stability of the reference genes among different tissues in normal crabs or after WSSV challenge was also tested by geNorm and NormFinder software. Among tissues, 18 S rRNA was most stably expressed in different tissues, followed by cyclophilin A and EF1-α, compared to β-actin and GAPDH. Upon to viral simulation, GAPDH was found to be the most stable internal control gene in gills and cyclophilin A was ranked as the most stable gene in hemocytes.

Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.

Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa).

BackgroundThe veined rapa whelk Rapana venosa is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in R. venosa. For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in R. venosa for use as internal controls for qRT-PCR.MethodsIn this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1α (EF-1α), α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1α subcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms.ResultsOf the 13 candidate genes tested, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. For the normalization of a single specific tissue, we suggested that EF-1α and NDUFA7 are the best combination in gonad, as well as COX1 and RL28 for intestine, EF-1α and RL5 for kidney, EF-1α and COX1 for gill, EF-1α and RL28 for Leiblein and mantle, EF-1α, RL5, and NDUFA7 for liver, GAPDH, PPIA, and RL28 for hemocyte. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured, and COX1 and RL5 were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization, PPIA, RS25, and RL28 for stage 1, RL5 and RL28 for stage 2 and 5, RL28 and NDUFA7 for stage 3, and PPIA and TUBB for stage 4.DiscussionOur results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development of R. venosa in the future.

Identification of novel and robust internal control genes from Volvariella volvacea that are suitable for RT-qPCR in filamentous fungi.

The selection of appropriate internal control genes (ICGs) is a crucial step in the normalization of real-time quantitative PCR (RT-qPCR) data. Housekeeping genes are habitually selected for this purpose, despite accumulating evidence on their instability. We screened for novel, robust ICGs in the mushroom forming fungus Volvariella volvacea. Nine commonly used and five newly selected ICGs were evaluated for expression stability using RT-qPCR data in eight different stages of the life cycle of V. volvacea. Three different algorithms consistently determined that three novel ICGs (SPRYp, Ras and Vps26) exhibited the highest expression stability in V. volvacea. Subsequent analysis of ICGs in twenty-four expression profiles from nine filamentous fungi revealed that Ras was the most stable ICG amongst the Basidiomycetous samples, followed by SPRYp, Vps26 and ACTB. Vps26 was expressed most stably within the analyzed data of Ascomycetes, followed by HH3 and β-TUB. No ICG was universally stable for all fungal species, or for all experimental conditions within a species. Ultimately, the choice of an ICG will depend on a specific set of experiments. This study provides novel, robust ICGs for Basidiomycetes and Ascomycetes. Together with the presented guiding principles, this enables the efficient selection of suitable ICGs for RT-qPCR.

Evaluation of Suitable Internal Control Genes for RT-qPCR in Yak Mammary Tissue during the Lactation Cycle.

The yak is primarily found throughout the Tibetan high plateau and the surrounding mountainous area of south central Asia; among its others attributes, its milk is very important for the local population. A key concern in the field of yak research is the better understanding of which genes control the production and composition of milk. The most accurate and sensitive method for gene expression analysis is quantitative reverse transcription polymerase chain reaction (RT-qPCR). It is essential for reliable RT-qPCR to be able to the normalize the data using internal control genes (ICGs). However, it is critical to assess the reliability of the normalization by testing multiple ICGs. Our objective was to uncover a reliable normalization for RT-qPCR data obtained from yak mammary tissue during the lactation cycle. We assessed the reliability of 10 ICGs (ACTB, EIF6, GAPDH, LRP10, MRPL39, MRPS15, MTG1, RPS8, RPS23, and UXT) using geNorm. The analysis revealed that all of the tested ICGs can be considered to be reliable, but the use of the 6 most stable ICGs should be applied to yield a reliable normalization factor (NF). We compared the results of 3 target genes (CSN1S1, ESR1, and MYC) normalized using 6, 3, or 1 of the best ICGs. We did not observe overall differences between the 3 normalization strategies with the exception of 1 time point in MYC. The use of only a single ICG is not recommended; thus, we concluded that the calculation of the NF using the 3 best ICGs, MRPS15, RPS23, and UXT, is a reliable normalization strategy for RT-qPCR data obtained from yak mammary tissue during pregnancy and lactation. A dilution effect of the ICGs due to a large increase in the mRNA of abundantly expressed genes in bovine and porcine mammary tissue during the lactation cycle was previously observed. To test for the presence of a dilution effect in our study, we evaluated the pattern of non-normalized RT-qPCR data of ICGs from pregnancy to lactation and compared them with the total RNA concentration, milk yield, and non-normalized RT-qPCR data of 3 target genes. With a few exceptions, the non- normalized RT-qPCR data for the tested ICGs was significantly increased by lactation and had a positive correlation with total RNA and the non-normalized RT-qPCR data of CSN1S1. These data clearly indicated the presence of a “concentration effect” of single mRNA that remains unexplained but needs to be accounted for during the normalization of RT-qPCR data. Based on our findings, we recommend that the NF of the MRPS15, RPS23, and UXT genes should be used in the normalization of RT-qPCR data obtained from mammary tissue of lactating yaks during pregnancy and lactation.

Identification and analysis of house-keeping and tissue-specific genes based on RNA-seq data sets across 15 mouse tissues

Recently, RNA-seq has become widely used technology for transcriptome profiling due to its single-base accuracy and high-throughput speciality. In this study, we applied a computational approach on an integrated RNA-seq dataset across 15 normal mouse tissues, and consequently assigned 8408 house-keeping (HK) genes and 2581 tissue-specific (TS) genes among UCSC RefGene annotation. Apart from some basic genomic features, we also performed expression, function and pathway analysis with clustering, DAVID and Ingenuity Pathway Analysis, indicating the physiological connections (tissues) and diverse biological roles of HK genes (fundamental processes) and TS genes (tissue-corresponding processes). Moreover, we used RT-PCR method to test 18 candidate HK genes and finally identified a novel list of highly stable internal control genes: Ywhae, Ddb 1, Eif4h, etc. In summary, this study provides a new HK gene and TS gene resource for further genetic and evolution research and helps us better understand morphogenesis and biological diversity in mouse.

Characterisation and Validation of House Keeping Gene for Expression Analysis in Catla catla (Hamilton)

A stable internal control gene or house keeping gene (HKG) is often used to normalise mRNA levels in different samples for expression analysis. In the present study, the authors identified and evaluated three HKGs, beta actin (β-actin), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor 1 alpha (EF1α) for gene expression study in Catla (Catla catla). Gene expression levels were quantified by quantitative real-time reverse transcription polymerase chain reaction in different tissues (liver, kidney, intestine, gill, muscle and brain) and developmental stages (0, 3, 6, 12, 24 h, and 5, 7, 9 days post-fertilization). Expression stability was evaluated by comparing the coefficients of variation of the cycle threshold values and stability index. All the tested HKGs exhibited wide expression range. The results showed that β-actin is the most stable gene followed by the EF1α and GAPDH in different tissues whereas GAPDH was most stable gene followed by EF1α and β-actin during embryonic development.

Real-time qPCR identifies suitable reference genes for Borna disease virus-infected rat cortical neurons.

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most commonly-used technique to identify gene expression profiles. The selection of stably expressed reference genes is a prerequisite to properly evaluating gene expression. Here, the suitability of commonly-used reference genes in normalizing RT-qPCR assays of mRNA expression in cultured rat cortical neurons infected with Borna disease virus (BDV) was assessed. The expressions of eight commonly-used reference genes were comparatively analyzed in BDV-infected rat cortical neurons and non-infected control neurons mainly across 9 and 12 days post-infection. These reference genes were validated by RT-qPCR and separately ranked by four statistical algorithms: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. Then, the RankAggreg package was used to construct consensus rankings. ARBP was found to be the most stable internal control gene at Day 9, and ACTB at Day 12. As the assessment of the validity of the selected reference genes confirms the suitability of applying a combination of the two most stable references genes, combining the two most stable genes for normalization of RT-qPCR studies in BDV-infected rat cortical neurons is recommended at each time point. This study can contribute to improving BDV research by providing the means by which to obtain more reliable and accurate gene expression measurements.

Reference gene selection for gene expression analysis in melon infected by Fusarium oxysporum f.sp. melonis

Eleven candidate reference genes were evaluated in stem tissues of two melon (Cucumis melo L) genotypes infected with Fusarium oxysporum f.sp. melonis (FOM) race 1, to investigate the most stable internal control gene to use in gene expression study in melon under biotic stress. The candidate reference genes, utilized in the qRT-PCR, were selected among the most common reference genes reported in melon literature as well as on melon sequences found in available databases. The expression stability of the reference genes selected, was tested by using the geNorm and NormFinder statistical algorithms. Irrespective of the plant response to FOM infection, there was substantial agreement in gene ranks of both software. The ribosomal protein L2 gene had the high transcript stability while the elongation factor, cyclophilin7 and 18S rRNA were the least stable. The best three reference genes, ribosomal protein L2, actin* and cyclophilin were used to normalize the expression data of the glutathione S-transferase in the resistant and susceptible plants. Differences in the expression data emerged for both genotypes and were more evident in the resistant one. The ribosomal protein L2 showed similar normalized expression values in both genotypes and thus it could be indicated as unique internal control for gene expression analysis in melon under FOM infection. This work represents the first in-depth study on reference genes validation in melon under biotic stress condition. The identification of such genes would improve the accuracy and reliability of gene expression studies and speed up candidate disease resistant gene analysis in melon.

β-2 microglobulin is unsuitable as an internal reference gene for the analysis of gene expression in human colorectal cancer

It is well-known that gene expression levels should be normalized to a carefully selected and appropriately stable internal control gene. However, numerous studies have demonstrated that the expression of housekeeping (HK) genes, typically used as internal control genes varies considerably. A number of studies have shown that β-2 microglobulin (B2M), an HK gene, frequently used as an internal reference gene, is expressed at low levels in colorectal cancer tissue, when assessed using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Due to the fact that the expression levels of various HK genes vary depending on the tissue type or experimental conditions, it has been suggested that several control genes should be analyzed in parallel for certain tissues. In the present study, mRNA expression levels of toll-like receptors 2 (TLR2) and 4 (TLR4) in sporadic human colorectal cancerous and non-cancerous tissues were analyzed relative to three HK genes, β-glucuronidase (GUS), β-actin (BA) and B2M, using a commercially available tool. Relative expression levels were quantified using the three genes individually and together, and TLR2 as well as TLR4 expression was compared in cancerous and non-cancerous colorectal tissue specimens. Consistent data were obtained in most cases when GUS and BA were used as internal control genes. When B2M was used as the internal control gene, TLR2 and TLR4 expression was demonstrated to be higher in cancerous compared to non-cancerous colorectal tissues. These results were consistent with previous observations of low-level B2M expression in cancerous colorectal tissue and suggest that B2M may be inappropriate as an internal control gene for gene expression studies of colorectal cancer.

Critical selection of internal control genes for quantitative real-time RT-PCR studies in lipopolysaccharide-stimulated human THP-1 and K562 cells

The choice of internal control genes is important since it may affect the study outcome in RT-qPCR. Indeed, it is well-known that expression levels of traditional internal control genes can vary across tissue types and across experimental settings within one specific tissue type. The aim of this study is an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in vitro different cultured cells, THP-1 and K562. The transcriptional stability of eleven potential internal control genes (RPL37A, ACTB, GAPDH, B2M, PPIB, PGK1, PPIA, SDHA, TBP, HPRT1 and RPL13A) were evaluated using RT-qPCR and were compared in different treatment, that was un-stimulated or LPS-stimulated cells. The raw Ct values were determined for each candidate gene at different time points following LPS-stimulated or unstimulated cells. Furthermore, all data were analyzed by the geNorm, BestKeeper, and NormFinder validation programs. Results indicated that PPIB and PGK1 were the most stable internal control genes in this study. RPL13A was found to be the least stable. This study provides the comprehensive reported assessment of internal control genes for use in expression studies in vitro cultured cells. These findings further emphasize the need to accurately validate candidate internal control genes in the study before use in gene expression studies using RT-qPCR.

Short Communication Suitable internal control microRNA genes for measuring miRNA abundance in pig milk during different lactation periods

Determination of an optimal set/number of internal control microRNA (miRNA) genes is a critical, but often undervalued, detail of quantitative gene expression analysis. No validated internal genes for miRNA quantitative PCR (q-PCR) in pig milk were available. We compared the expression stability of six porcine internal control miRNA genes in pig milk from different lactation periods (1 h, 3 days, 7 days, 14 days, 21 days, and 28 days postpartum), using an EvaGreen q-PCR approach. We found that using the three most stable internal control genes to calculate the normalization factor is sufficient for producing reliable q-PCR expression data. We also found that miRNAs are superior to ribosomal RNA (rRNA) and snRNA, which are commonly used as internal controls for normalizing miRNA q-PCR data. In terms of economic and experimental feasibility, we recommend the use of the three most stable internal control miRNA genes (miR-17, -107 and -103) for calculating the normalization factors for pig milk samples from different lactation periods. These results can be applied to future studies aimed at measuring miRNA abundance in porcine milk.

Validation of reference genes for real-time qRT-PCR normalization during cold acclimation in Eucalyptus globulus

During the last few years, many studies have directed their efforts at elucidating the molecular mechanisms that regulate plant response to cold stress using gene expression analysis. Quantitative real-time qRT-PCR has great advantages compared to traditional transcriptional detection methods due to its high sensibility, reproducibility, and specificity for the detection of low quantities of RNA. However, this technique requires the use of one or several housekeeping genes. In this work, the expression stabilities of six housekeeping genes (EF1α, ACT, α-TUB, PDF, SAND, and UBC) during the cold acclimation of E. globulus plants was analyzed. An ELIP gene that responds to photooxidative stress caused by light and cold stress was used as the target gene to identify the most suitable internal control for normalizing real-time qRT-PCR. Two additional genes involved in the ABA biosynthesis pathway (NCED) and sugar metabolism (GS) were analyzed with the most stable internal control genes in order to check the results found with the ELIP gene. The expressions of UBC, α-TUB and EF1α were the most stable across acclimation and de-acclimation treatments. The expressions of the other housekeeping genes tested varied depending upon the conditions. The relative quantification of ELIP changed according to identities and the number of reference genes used, thus demonstrating the importance of selecting an appropriate number of reference genes in order to achieve an accurate and reliable normalization of gene expression during cold acclimation in E. globulus.