TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells

장시정,Jeon Ryoung-Hoon,Kim Hwan-Deuk,Hwang Jong-Chan,Lee Hyeon-Jeong,배슬기,Lee Sung-Lim,노규진,김승준,Lee Won-Jae 아세아·태평양축산학회 2020 Animal Bioscience Vol.33 No.12

Objective: Quantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs. Methods: The cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins. Results: Based on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance. Conclusion: This study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs.

Evaluation of reference genes for quantitative real-time PCR to investigate seasonal and labor-specific expression profiles of the honey bee abdomen

Moon, KyungHwan,Lee, Si Hyeock,Kim, Young Ho 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.2 No.1

<P><B>Abstract</B></P> <P>Owing to the highly developed sociality, division of labor, and passive population management of their colonies, honey bees, <I>Apis mellifera</I> L., have been widely used as model insects to investigate the physiological roles of the genes putatively involved in social development in insects. Quantitative real-time polymerase chain reaction (qRT-PCR) has been broadly applied to investigate the expression patterns of such target genes. However, the selection of suitable reference genes is an essential step to ensure the accurate quantification of target gene transcription levels with this method. Therefore, in this study, we selected seven potential reference genes (<I>rp49, rpL32, rpS18, tbp, tub</I>, <I>gapdh,</I> and <I>ace2</I>) and determined their PCR efficiencies. The seasonal expression efficiencies of five genes (<I>rpL32, rpS18, tbp</I>, <I>gapdh</I>, and <I>ace2</I>) that displayed 90% to 110% amplification efficiencies were then evaluated using three software packages (geNorm, NormFinder, and BestKeeper), and were also applied in the normalization of seasonal expression patterns of a target gene (<I>vg</I>) in the abdomens of forager and nurse honey bee workers. The three software packages resulted in slightly different gene stability ranks, but overall the combination of two genes (<I>rpS18</I> and <I>gapdh</I>) was determined to be the most optimal for use in the normalization of target gene expression in forager. However, a single gene (either <I>rpL32</I> or <I>rpS18</I>) should be applied in the nurse. In the comparison between foragers and nurses, either <I>rpL32</I>, <I>rpS18</I>, or <I>gapdh</I>, is suggested to be used as the reference gene for qRT-PCR-based determination of seasonal and labor-specific gene expression profiles.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Reference genes for qRT-PCR assay were validated in the honey bee abdomen. </LI> <LI> We evaluated stability of seasonal and labor-dependent reference gene expression. </LI> <LI> The combination of <I>rpS18</I> and <I>gapdh</I> is required for gene normalization in foragers. </LI> <LI> A single gene, either <I>rpS18</I> or <I>gapdh</I>, can be used as the reference in nurses. </LI> <LI> Either <I>rpS18, rpL32</I>, or <I>gapdh</I> can be used in comparisons of the two worker types. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Evaluation and validation of reference genes for SYBR Green qRT-PCR normalization in Sesamia inferens (Lepidoptera: Noctuidae)

Yanhui Lu,Xu-Song Zheng,Qi Liang,Hong-Xing Xu,Yajun Yang,Junce Tian,Xiaochan He,Zhong-Xian Lu 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.4

The pink stemborer, Sesamia inferens (Walker) (Lepidoptera: Noctuidae), has gradually become amajor threat to rice production in Asian rice growth area. Using reverse-transcription quantitative real-time PCR (qRT-PCR) to investigate gene expression of S. inferens under different conditions is a popular technique. However, numerous studies demonstrate that the experimental conditions affect the stability of reference genes. Thus, it is necessary to normalize qRT-PCR data under a specific condition using a stable reference gene as an internal control. In the present study, five algorithms (Delta Ctmethod, Bestkeeper, geNorm,Normfinder, and RefFinder) and seven candidate reference genes were evaluated to find out the most suitable reference genes under different biotic and abiotic conditions including different tissues and developmental stages, exposure to insecticides, different rice varieties, and different diets. Our results showed that the more stable series of reference genes were as follows: GAPDH, RPL10, and RPS3 for different tissues;GAPDH and RPS3 for developmental stages; RPS3 and EF1 for larval insecticide exposure; RPS3 and RPL10 for larvae reared on different rice varieties; RPS3 and ACTB for larvae fed on different diets. Hence, a combination of the best sets of reference genes for normalization of expression analysis will increase the accuracy and reliability of qRT-PCR data in S. inferens. In addition, these results establish the basis for selection of reference genes in other lepidopterous insect species.

Selection and evaluation of reference genes for gene expression using quantitative real‐time PCR in Mythimna separata walker (Lepidoptera: Noctuidae)

Bai‐Zhong Zhang,Jun-Jie LIU,Xi-Ling CHEN,Guo-Hui YUAN 한국곤충학회 2018 Entomological Research Vol.48 No.5

In order to precisely assess gene expression levels, the suitable internal reference genes must be served to quantify real‐time reverse transcription polymerase chain reaction (RT‐qPCR) data. For armyworm, Mythimna separata, which reference genes are suitable for assessing the level of transcriptional expression of target genes have yet to be explored. In this study, eight common reference genes, including β‐actin (β‐ACT), 18 s ribosomal (18S), 28S ribosomal (28S), glyceraldehyde‐3‐phosphate (GAPDH), elongation fator‐alpha (EF1α), TATA box binding protein (TBP), ribosomal protein L7 (RPL7), and alpha‐tubulin (α‐TUB) that in different developmental stages, tissues and insecticide treatments of M. separata were evaluated. To further explore whether these genes were suitable to serve as endogenous controls, three software‐based approaches (geNorm, BestKeeper, and NormFinder), the delta Ct method, and one web‐based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized HSP70, and MsepCYP321A10 gene expression data. We found that the most suitable reference genes for the different experimental conditions. For developmental stages, 28S/RPL7 were the optimal reference genes, both RPL7/EF1α were suitable for experiments of different tissues, whereas for insecticide treatments, 28S/α‐TUB were suitable for normalizations of expression data. In addition, 28S/α‐TUB were the suitable reference genes because they have the most stable expression among different developmental stages, tissues and insecticide treatments. Our work is the first report on reference gene selection in M. separata, and might serve as a precedent for future gene expression studies.

Normal Echocardiographic Measurements in a Korean Population Study: Part I. Cardiac Chamber and Great Artery Evaluation

최진오,신미승,김미정,정해억,박정랑,손일석,김형섭,박성미,유남진,최정현,김형관,조구영,이미래,박진선,심지영,김대희,신대희,신길자,신성희,김계훈,박재형,이상엽,김우식,박승우 한국심초음파학회 2015 Journal of Cardiovascular Imaging (J Cardiovasc Im Vol.23 No.3

Background: Measurement of the cardiac chamber is essential, and current guidelines recommend measuring and reportingvalues for both sides of the cardiac chamber during echocardiographic evaluation. Normal echocardiographic reference values havebeen suggested previously, but detailed information about right-sided chambers and values according to gender was not included. Methods: This is a prospective multicenter (23 centers) study evaluating normal Korean adult subjects using comprehensiveechocardiography. We included normal adult subjects (age; 20–79 years old) who had no significant cardiac disorders or illnesses, annusuchas hypertension or diabetes, which could affect cardiac structure and function. We measured the cardiac chamber includingboth right and left ventricles as well as atria according to current echocardiography guidelines and compared values according togender and age groups. Results: A total of 1003 subjects were evaluated and the mean age was 48 ± 16 years. Left ventricular (LV) dimensions increased,but LV volume decreased in older subjects. Right ventricular (RV) area decreased in women and older subjects, and theRV long-axis dimension showed a similar trend. Left atrial (LA) volume increased in men but there were no differences in LA volumeindex between men and women. The dimension of great arteries increased in men and older subjects. Conclusion: Since there were considerable differences between men and women and in the different age groups, and thetrends differed significantly between different echo variables, normal echocardiographic cutoff values should be differentially appliedbased on age and gender.

Establishment of Normal Reference Data of Analysis in the Fresh and Cryopreserved Canine Spermatozoa

박병준,이현정,이성림,노규진,김승준,이원재 사단법인 한국동물생명공학회 2018 한국동물생명공학회지 Vol.33 No.2

The cryopreservation has been extensively applied in many cells including spermatozoa (semen) during past several decades. Especially, the canine spermatozoa cryopreservation has contributed on generation of progeny of rare/genetically valuable dog breeds, genome resource banking and transportation of male germplasm at a distant place. However, severe and irreversible damages to the spermatozoa during cryopreservation procedures such as the thermal shock (cold shock), formation of intracellular ice crystals, osmotic shock, stress of cryoprotectants and generator of reactive oxygen species (ROS) have been addressed. According as a number of researches have been conducted to overcome these problems and to advance cryopreservation technique, several analytical methods have been employed to evaluate the quality of the fresh or cryopreserved canine spermatozoa in regards to the motility, morphology, integrity of membrane and DNA, mitochondrial activity, ROS generation, binding affinity to oocytes, in vitro fertilization potential and fertility potential by artificial insemination. Because the study designs with certain application of analytical methods are selective and varied depending on each experimental objective and laboratory condition, it is necessary to establish the normal reference data of the fresh or cryopreserved canine spermatozoa for each analytical method to monitor experimental procedure, to translate raw data and to discuss results. Here, we reviewed the recent articles to introduce various analytical methods for the canine spermatozoa as well as to establish the normal reference data for each analytical method in the fresh or cryopreserved canine spermatozoa, based on the results of the previous articles. We hope that this review contributes to the advancement of cryobiology in canine spermatozoa.

Normal Echocardiographic Measurements in a Korean Population Study: Part II. Doppler and Tissue Doppler Imaging

최진오,신미승,김미정,정해억,박정랑,손일석,김형섭,박성미,유남진,최정현,김형관,조구영,이미래,박진선,심지영,김대희,신대희,신길자,신성희,김계훈,박재형,이상엽,김우식,박승우 한국심초음파학회 2016 Journal of Cardiovascular Imaging (J Cardiovasc Im Vol.24 No.2

Background: Hemodynamic and functional evaluation with Doppler and tissue Doppler study as a part of comprehensiveechocardiography is essential but normal reference values have never been reported from Korean normal population especially accordingto age and sex. Methods: Using Normal echOcaRdiographic Measurements in a KoreAn popuLation study subjects, we obtained normal referencevalues for Doppler and tissue Doppler echocardiography including tricuspid annular velocities according to current guidelinesand compared values according to gender and age groups. Results: Mitral early diastolic (E) and late diastolic (A) velocity as well as E/A ratio were significantly higher in women comparedto those in men. Conversely, mitral peak systolic and late diastolic annular velocity in both septal and lateral mitral annuluswere significantly lower in women compared to those in men. However, there were no significant differences in both septal andlateral mitral early diastolic annular (e’) velocity between men and women. In both men and women, mitral E velocity and its decelerationtime as well as both E/A and E/e’ ratio considerably increased with age. There were no significant differences in tricuspidinflow velocities and tricuspid lateral annular velocities between men and women except e’ velocity, which was significantlyhigher in women compared to that in men. However, changes in both tricuspid inflow and lateral annular velocities according toage were similar to those in mitral velocities. Conclusion: Since there were significant differences in Doppler and tissue Doppler echocardiographic variables between menand women and changes according to age were even more considerable in both gender groups, normal Doppler echocardiographicvalues should be differentially applied based on age and sex.

Establishment of Normal Reference Data of Analysis in the Fresh and Cryopreserved Canine Spermatozoa

Byung-Joon Park,Seung-Joon Kim,Hyeon-Jeong Lee,Sung-Lim Lee,Gyu-Jin Rho,Won-Jae Lee 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.2

The cryopreservation has been extensively applied in many cells including spermatozoa (semen) during past several decades. Especially, the canine spermatozoa cryopreservation has contributed on generation of progeny of rare/genetically valuable dog breeds, genome resource banking and transportation of male germplasm at a distant place. However, severe and irreversible damages to the spermatozoa during cryopreservation procedures such as the thermal shock (cold shock), formation of intracellular ice crystals, osmotic shock, stress of cryoprotectants and generator of reactive oxygen species (ROS) have been addressed. According as a number of researches have been conducted to overcome these problems and to advance cryopreservation technique, several analytical methods have been employed to evaluate the quality of the fresh or cryopreserved canine spermatozoa in regards to the motility, morphology, integrity of membrane and DNA, mitochondrial activity, ROS generation, binding affinity to oocytes, in vitro fertilization potential and fertility potential by artificial insemination. Because the study designs with certain application of analytical methods are selective and varied depending on each experimental objective and laboratory condition, it is necessary to establish the normal reference data of the fresh or cryopreserved canine spermatozoa for each analytical method to monitor experimental procedure, to translate raw data and to discuss results. Here, we reviewed the recent articles to introduce various analytical methods for the canine spermatozoa as well as to establish the normal reference data for each analytical method in the fresh or cryopreserved canine spermatozoa, based on the results of the previous articles. We hope that this review contributes to the advancement of cryobiology in canine spermatozoa.

Normal 2-Dimensional Strain Values of the Left Ventricle: A Substudy of the Normal Echocardiographic Measurements in Korean Population Study

박재형,이주희,이상엽,최진오,신미승,김미정,정해억,박정랑,손일석,김형섭,박성미,유남진,최정현,김형관,조구영,이미래,박진선,심지영,김대희,신대희,신길자,신성희,김계훈,김우식,박승우 한국심초음파학회 2016 Journal of Cardiovascular Imaging (J Cardiovasc Im Vol.24 No.4

Background: It is important to understand the distribution of 2-dimensional strain values in normal population. We performeda multicenter trial to measure normal echocardiographic values in the Korean population. Methods: This was a substudy of the Normal echOcardiogRaphic Measurements in KoreAn popuLation (NORMAL) study. Echocardiographic specialists measured frequently used echocardiographic indices in healthy people according to a standardizedmethod at 23 different university hospitals. The strain values were analyzed from digitally stored images. Results: Of a total of 1003 healthy participants in NORMAL study, 2-dimensional strain values were measured in 501 subjects(265 females, mean age 47 ± 15 years old) with echocardiographic images only by GE echocardiographic machines. Interventricularseptal thickness, left ventricular (LV) posterior wall thickness, systolic and diastolic LV dimensions, and LV ejectionfraction were 7.5 ± 1.0 mm, 7.4 ± 1.0 mm, 29.9 ± 2.8 mm, 48.9 ± 3.6 mm, and 62 ± 4%, respectively. LV longitudinal systolicstrain (LS) values of apical 4-chamber (A4C) view, apical 3-chamber (A3C) view, apical 2-chamber (A2C) view, and LV global LS(LVGLS) were -20.1 ± 2.3, -19.9 ± 2.7, -21.2 ± 2.6, and -20.4 ± 2.2%, respectively. LV longitudinal systolic strain rate (LVLSR)values of the A4C view, A3C view, A2C view, and LV global LSR (LVGLSR) were -1.18 ± 0.18, -1.20 ± 0.21, -1.25 ± 0.21, and-1.21 ± 0.21-s, respectively. Females had lower LVGLS (-21.2 ± 2.2% vs. -19.5 ± 1.9%, p < 0.001) and LVGLSR (-1.25 ± 0.18-svs. -1.17 ± 0.15-s, p < 0.001) values than males. Conclusion: We measured LV longitudinal strain and strain rate values in the normal Korean population. Since considerablegender differences were observed, normal echocardiographic cutoff values should be differentially applied based on sex.

Establishment of Normal Reference Data of Analysis in the Fresh and Cryopreserved Canine Spermatozoa

Park, Byung-Joon,Lee, Hyeon-Jeong,Lee, Sung-Lim,Rho, Gyu-Jin,Kim, Seung-Joon,Lee, Won-Jae The Korean Society of Embryo Transfer 2018 한국동물생명공학회지 Vol.33 No.2

The cryopreservation has been extensively applied in many cells including spermatozoa (semen) during past several decades. Especially, the canine spermatozoa cryopreservation has contributed on generation of progeny of rare/genetically valuable dog breeds, genome resource banking and transportation of male germplasm at a distant place. However, severe and irreversible damages to the spermatozoa during cryopreservation procedures such as the thermal shock (cold shock), formation of intracellular ice crystals, osmotic shock, stress of cryoprotectants and generator of reactive oxygen species (ROS) have been addressed. According as a number of researches have been conducted to overcome these problems and to advance cryopreservation technique, several analytical methods have been employed to evaluate the quality of the fresh or cryopreserved canine spermatozoa in regards to the motility, morphology, integrity of membrane and DNA, mitochondrial activity, ROS generation, binding affinity to oocytes, in vitro fertilization potential and fertility potential by artificial insemination. Because the study designs with certain application of analytical methods are selective and varied depending on each experimental objective and laboratory condition, it is necessary to establish the normal reference data of the fresh or cryopreserved canine spermatozoa for each analytical method to monitor experimental procedure, to translate raw data and to discuss results. Here, we reviewed the recent articles to introduce various analytical methods for the canine spermatozoa as well as to establish the normal reference data for each analytical method in the fresh or cryopreserved canine spermatozoa, based on the results of the previous articles. We hope that this review contributes to the advancement of cryobiology in canine spermatozoa.