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Selective inhibition of genes in methicillin resistant Staphylococcus aureus (MRSA) treated with Melastoma malabathricum methanol extract

Zulaikah Mohamed, Nazlina Ibrahim, Ismail Ahmad

Sains Malaysiana, 2008;37(1):107-113.

Methanol extract of Melastoma malabathricum leaves inhibited the growth of Staphylococcus aureus and six clinical isolates of Methicilin Resistant Stapyhlococcus aureus (MRSA 1-6). The minimum inhibitory concentration (MIC) of test substance was 1.565mg/ml and the minimum bacteriocidal concentration (MBC) was 3.125 mg/ml. The methanol extract suppressed RNA synthesis at 10 mg/ml as shown by RNA profile which was devoid of three bands compared to the control. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using seven primer pairs was only successful in amplifying four cDNA amplicons. The failure to amplify three cDNA amplicons for three primer pairs corresponding to gyrA, femA and nuc genes, implied the possibility of suppression of the corresponding mRNA. Electrophoretic separation of endogenous and exogenuos bacterial proteins showed that three and five protein, respectively were not expressed. One endogenous and three exogenous proteins were over-expressed in treated MRSA compared with untreated control. The results of the molecular and proteomic analyses are in agreement, and based on primers being used, methanol extract of M. malabathricum leaves possibly inhibits MRSA growth through inhibition of DNA synthesis, peptidoglycan production, and nuclease production.
Keywords: Methicillin resistant Staphylococcus aureus; Melastoma malabathricum; gene expression; protein production

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Development of a human salivary gland organoid model

Zulaiha A. Rahman, Colin D. Bingle, Lynne Bingle

Malaysian Journal of Medicine and Health Sciences, 2019;15(107):1-1.
MyJurnal

Introduction: Currently, organoid technology provides a useful tool for modelling human organ development and pathologies in vitro. Salivary gland (SG) organoids developed from mice SG cells display self-organizing properties closely mimic the native organ. Thus, this study would like to investigate the potential of this organoid system to de-velop a human salivary gland in vitro. Methods: Organoids were developed from biopsy samples of normal human sublingual gland tissue. Cells were isolated and cultured in Matrigel at an Air Liquid Interface (ALI) for up to 14 days in an enriched media supplementing with Wnt-3A, R-spondin1, EGF, and FGF2. Specific differentiation factors like TGFβ, BMP, and LIMK inhibitors were added to enriched media for further differentiation studies. Haematoxylin and eosin-stained sections of the cultures were used to visualise growth. RT-PCR, immunohistochemistry and immunoflu-orescence were used to determine the differential expression of cell-specific markers. Results: Human SG organoids developed when the cells were grown in Matrigel at ALI in a defined culture system. The addition of TGFβ inhibitor and all the inhibitors (TGFβ, BMP and LIMK) to the culture media affected SG organoids development by displaying distinct characteristics that closely resemble native glands and expressed specific cell-type markers; BPIFA2, AQP5, CK5 and E-cadherin. The inhibition of BMP signalling demonstrated SG organoids growth more into ductal-like struc-tures and expressed ductal cell marker, CK7. While LIM kinase inhibition signalling showed significantly higher of amylase activity assay. Conclusion: This study certainly offers valuable insight into determining the optimal culture conditions for developing human SG organoids.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei

Zokaeifar H, Balcázar JL, Saad CR, Kamarudin MS, Sijam K, Arshad A, et al.

Fish Shellfish Immunol, 2012 Oct;33(4):683-9.
PMID: 22659618 DOI: 10.1016/j.fsi.2012.05.027

We studied the effect of two probiotic Bacillus subtilis strains on the growth performance, digestive enzyme activity, immune gene expression and disease resistance of juvenile white shrimp (Litopenaeus vannamei). A mixture of two probiotic strains, L10 and G1 in equal proportions, was administered at two different doses 10(5) (BM5) and 10(8) (BM8) CFU g(-1) feed to shrimp for eight weeks. In comparison to untreated control group, final weight, weight gain and digestive enzyme activity were significantly greater in shrimp fed BM5 and BM8 diets. Significant differences for specific growth rate (SGR) and survival were recorded in shrimp fed BM8 diet as compared with the control; however, no significant differences were recorded for food conversion ratio (FCR) among all the experimental groups. Eight weeks after the start of the feeding period, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 63.3%, whereas cumulative mortality of the shrimp that had been given probiotics was 20.0% with BM8 and 33.3% with BM5. Subsequently, real-time PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was significantly up-regulated (P

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/veterinary
Fulltext Molecular characterization of variant infectious bronchitis virus in China, 2019: Implications for control programmes

Zhang X, Deng T, Lu J, Zhao P, Chen L, Qian M, et al.

Transbound Emerg Dis, 2020 May;67(3):1349-1355.
PMID: 31943814 DOI: 10.1111/tbed.13477

Infectious bronchitis virus (IBV), an ongoing emergence enveloped virus with a single-stranded positive-sense RNA genome, belongs to the Gammacoronavirus genus in the Coronaviridae family. IBV-associated tracheitis, nephritis, salpingitis, proventriculitis and egg drop have caused devastating economic losses to poultry industry worldwide. Since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with IBV in China. Here, we described two IB outbreaks with severe respiratory system or kidney injury in IBV-vaccinated commercial poultry farms in central China. Other possible causative viral pathogens, including avian influenza virus (AIV), Newcastle disease virus (NDV) and Kedah fatal kidney syndrome virus (KFKSV), were excluded by reverse transcription-polymerase chain reaction (RT-PCR), and three virulent IBV strains, HeN-1/China/2019, HeN-2/China/2019 and HeN-101/China/2019, were identified. Although the gross pathologic appearance of these two IB outbreaks was different, the newly identified IBV strains were all closely related to the ck/China/I0529/17 strain and grouped into GI-19 genotype clade based on the sequencing and phylogenetic analysis of the complete S1 genes. Moreover, there are still some evolutionary distance between the newly identified IBV strains, HeN-101/China/2019 in particular, and other GI-19 strains, suggesting that Chinese IBV strains constantly emerge and evolve towards different directions. In conclusion, this study provided an insight of the recently emerging IBV outbreaks in IBV-vaccinated commercial poultry farms and identified the genetic characteristics of three virulent GI-19 IBV strains, which shows the need to carry out proper preventive measures and control strategies.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Preliminary evaluation of various rapid influenza diagnostic test methods for the detection of the novel influenza A (H1N1) in Universiti Kebangsaan Malaysia Medical Centre

Zetti ZR, Wong KK, Haslina M, Ilina I

Med J Malaysia, 2010 Mar;65(1):27-30.
PMID: 21265244 MyJurnal

We evaluated the performance of four rapid influenza diagnostic test methods (RIDT) compared to real-time reverse-transcription polymerase chain reaction (rRT-PCR), for the detection of the novel swine-origin influenza A (H1N1) virus (S-OIV) in August 2009. A total of 270 respiratory specimens were tested with rRT-PCR, where 74 of these were tested by BinaxNow (Inverness), 80 by QuickVue (Quidel), 37 by Influenza A Antigen Rapid Test (Rockeby Biomed) and 79 by Directigen (BD). The sensitivities ranged from 4.4% to 37.0%, specificities 90.9% to 100.0%, positive predictive values 75.0% to 100.0% and negative predictive values 32.3% to 75.0%. RIDT were able to detect S-OIV but the sensitivities were low. The limitations of RIDT must be considered when interpreting results for clinical management.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Multiplex real-time RT-PCR assay for diagnosis of Dengue, Zika and Chikungunya infections

Zarina Mohd Zawawi, Tengku Rogayah Tengku Abdul Rashid, Amir Hussien Adiee, Murni Maya Sari, Ravindran Thayan

Malaysian Journal of Medicine and Health Sciences, 2019;15(106):5-5.
MyJurnal

Introduction: Dengue virus (DENV), Zika virus (ZIKV) and Chikungunya virus (CHIKV) are Arboviruses that are transmitted by the same vector, Aedes aegypti. Dengue has become a global problem since the Second World War and is common in more than 110 countries. In Malaysia, dengue is a major disease burden as total economic costs to the country as a result of dengue is close to RM1.05 billion in 2010 and estimated to rise to 1.3 billion by 2020. Apart from Dengue, Zika and Chikungunya are the other important mosquito borne diseases in Malaysia. The aim of this study was to develop a multiplex real-time assay for simultaneous detection of DENV, ZIKV and CHIKV in clinical specimens. Methods: The published singleplex protocols were used with key modifications to implement a triplex assay. A one-step multiplex real-time RT-PCR assay was developed that can simultaneously detect RNA of DENV, ZIKV and CHIKV with good performance for a routine diagnostic use. The assay was evaluated for inter- and intra-reproducibility by mean CT value. The diagnostic sensitivity was tested with 135 archived samples which had been defined positive or negative by routine singleplex assays. Whole blood, plasma and urines were used in this study. Results: Intra- and inter-reproducibility and sensitivity varied from 0.10% to 4.73% and from 0.45% to 5.98% for each virus respectively. The specificity of detection was 100%. The multiplex real-time RT-PCR assay showed concordance with test results performed by routine singleplex assays. No cross reaction was observed for any of the clinical samples. Conclusion: The development of a rapid, sensitive and specific molecular assay for DENV, ZIKV and CHIKV infections will produce a greater diagnostic capacity in our laboratory. This multiplex approach is cost effective and robust with the concurrent detection of 3 viruses of public health concern.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Antiviral activity of four types of bioflavonoid against dengue virus type-2

Zandi K, Teoh BT, Sam SS, Wong PF, Mustafa MR, Abubakar S

Virol J, 2011;8:560.
PMID: 22201648 DOI: 10.1186/1743-422X-8-560

Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic concentration 50 (CC50) to inhibitory concentration 50 (IC50) for each compound.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Stemness gene expression profile of human adipose derived stem cells in long-term culture

Zaman WS, Makpol S, Santhapan S, Chua KH

Med J Malaysia, 2008 Jul;63 Suppl A:61-2.
PMID: 19024984

It is crucial to know whether stem cells retain its stemnness properties after advance in vitro manipulation. The objective of this study was to investigate the stemness gene expression of human adipose tissue derived stem cells (ADSCs) in long-term culture using quantitative RT-PCR technique. Our data showed that the expression level of Sox-2, Rex-1, FGF-4, Nanog, Nestin, BST-1, FZD-9 and Oct-4 were decreased gradually in long-term culture. This could mean that the ability of ADSCs to differentiate into other cell lineages reduce after extensive culture.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Molecular detection of the New Delhi metallo-β-lactamase-1 gene in Enterobacteriaceae isolates in a tertiary medical centre

Zainol Abidin NZ, Sulong A, Alfizah H, Muttaqillah NA, Ding CH

Malays J Pathol, 2015 Dec;37(3):227-32.
PMID: 26712667 MyJurnal

New Delhi metallo-β-lactamase-1 (NDM-1) is a relatively recent carbapenemase enzyme that inactivates all β-lactam antibiotics with the exception of aztreonam. This study aims to ascertain the baseline prevalence and antibiotic susceptibility patterns of NDM-1-producing Enterobacteriaceae in a tertiary medical center in Malaysia.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Differentiation of dental pulp stem cells into neuron-like cells in serum-free medium

Zainal Ariffin SH, Kermani S, Zainol Abidin IZ, Megat Abdul Wahab R, Yamamoto Z, Senafi S, et al.

Stem Cells Int, 2013;2013:250740.
PMID: 24348580 DOI: 10.1155/2013/250740

Dental pulp tissue contains dental pulp stem cells (DPSCs). Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells) are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146(+) , Cd166(+) , and Cd31(-) in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext In vitro chondrogenesis transformation study of mouse dental pulp stem cells

Zainal Ariffin SH, Kermani S, Megat Abdul Wahab R, Senafi S, Zainal Ariffin Z, Abdul Razak M

ScientificWorldJournal, 2012;2012:827149.
PMID: 22919354 DOI: 10.1100/2012/827149

A major challenge in the application of mesenchymal stem cells in cartilage reconstruction is that whether the cells are able to differentiate into fully mature chondrocytes before grafting. The aim of this study was to isolate mouse dental pulp stem cells (DPSC) and differentiate them into chondrocytes. For this investigation, morphological, molecular, and biochemical analyses for differentiated cells were used. To induce the chondrocyte differentiation, DPSC were cultured in chondrogenic medium (Zen-Bio, Inc.). Based on morphological analyses using toluidine blue staining, proteoglycan products appear in DPSC after 21 days of chondrocyte induction. Biochemical analyses in differentiated group showed that alkaline phosphatase activity was significantly increased at day 14 as compared to control (P < 0.05). Cell viability analyses during the differentiation to chondrocytes also showed that these cells were viable during differentiation. However, after the 14th day of differentiation, there was a significant decrease (P < 0.05) in the viability proportion among differentiated cells as compared to the control cells. In RT-PCR molecular analyses, mouse DPSC expressed Cd146 and Cd166 which indicated that these cells belong to mesenchymal stem cells. Coll I and Coll II markers showed high expression after 14 and 21 days, respectively. In conclusion, this study showed that DPSC successfully differentiated into chondrocytes.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Identifications of drug resistance mutations among antiretroviral treatment naive HIV-1 patients in Peninsular Malaysia

Zain RM, Ibrahim N, Ismail S, Suppiah J, Mat Rahim NA, Thayan R

Asian Pac J Trop Med, 2017 Jan;10(1):75-78.
PMID: 28107870 DOI: 10.1016/j.apjtm.2016.12.005

OBJECTIVE: To determine drug resistance mutations and the HIV-1 subtypes among antiretroviral treatment naive HIV-1 patients in Peninsular Malaysia.
METHODS: A total of 45 samples from four hospitals that provide HIV viral load services were subjected to the amplification of the protease and two third of reverse transcriptase regions of the pol gene by RT-PCR and Sanger sequencing. Drug resistance mutation (DRM) interpretation reports the presence of mutations related to protease inhibitors (PIs), Nucleoside reverse-transcriptase inhibitors (NRTI) and Non-nucleoside reverse-transcriptase inhibitors (NNRTI) based on analysis using Stanford HIV database program.
RESULTS: DRMs were identified in 35% of patients, among which 46.7% of them showed minor resistance to protease inhibitor with A71V and L10l were the commonest DRMs detected. About 21.4% and 50.0% of patients had mutations to NRTIs and NNRTIs, respectively. CRF01_AE was found to be the predominant HIV-1 subtype.
CONCLUSIONS: These findings have served as an initial crucial data in determining the prevalence of transmitted HIV-1 drug resistance for the country. However, more samples from various parts of the country need to be accumulated and analyzed to provide overall HIV-1 drug resistance in the country.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus)

Yusuf NH, Ong WD, Redwan RM, Latip MA, Kumar SV

Gene, 2015 Oct 15;571(1):71-80.
PMID: 26115767 DOI: 10.1016/j.gene.2015.06.050

MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Nucleic Acid-Based Diagnostic Tests for the Detection SARS-CoV-2: An Update

Yu CY, Chan KG, Yean CY, Ang GY

Diagnostics (Basel), 2021 Jan 01;11(1).
PMID: 33401392 DOI: 10.3390/diagnostics11010053

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began as a cluster of pneumonia cases in Wuhan, China before spreading to over 200 countries and territories on six continents in less than six months. Despite rigorous global containment and quarantine efforts to limit the transmission of the virus, COVID-19 cases and deaths have continued to increase, leaving devastating impacts on the lives of many with far-reaching effects on the global society, economy and healthcare system. With over 43 million cases and 1.1 million deaths recorded worldwide, accurate and rapid diagnosis continues to be a cornerstone of pandemic control. In this review, we aim to present an objective overview of the latest nucleic acid-based diagnostic tests for the detection of SARS-CoV-2 that have been authorized by the Food and Drug Administration (FDA) under emergency use authorization (EUA) as of 31 October 2020. We systematically summarize and compare the principles, technologies, protocols and performance characteristics of amplification- and sequencing-based tests that have become alternatives to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. We highlight the notable features of the tests including authorized settings, along with the advantages and disadvantages of the tests. We conclude with a brief discussion on the current challenges and future perspectives of COVID-19 diagnostics.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Rapid detection and serotyping of dengue virus by multiplex RT-PCR and real-time SYBR green RT-PCR

Yong YK, Thayan R, Chong HT, Tan CT, Sekaran SD

Singapore Med J, 2007 Jul;48(7):662-8.
PMID: 17609830

Dengue fever and dengue haemorrhagic fever currently rank highly among the newly-emerging infectious diseases, and are considered to be the most important arboviral disease worldwide. The definitive diagnosis is culture analysis, but practical considerations limit its use. Also, the period for viral detection is limited. Within a day or two after fever subsides, rising levels of antibodies interfere with viral cultures. An alternative to this quandary is the use of viral RNA detection assays. In our laboratory, a reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed using a set of degenerate primers.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
A modified in situ RT-PCR method for localizing fungal-specific gene expression in Candida-infected mice renal cells

Yong VC, Ong KW, Sidik SM, Rosli R, Chong PP

J Microbiol Methods, 2009 Nov;79(2):242-5.
PMID: 19737582 DOI: 10.1016/j.mimet.2009.08.019

In situ Reverse Transcriptase PCR (in situ RT-PCR) can amplify mRNA and localize gene expression in cells. However, this method is not feasible in fungi as the thick fungal cell wall constitutes a barrier to this procedure. We developed a two step in situ RT-PCR procedure which enabled the detection and localization of Candida tropicalis mRNA expression in formalin-fixed, paraffin-embedded (FFPE) mouse kidney sections. This in situ hybridization study revealed the first direct evidence for deposition of Candida tropicalis secreted aspartic proteinase 2 (CtSAP2) in the tip of pseudohyphae and its involvement in acute systemic candidiasis. We conclude that in situ RT-PCR can be successfully applied to FFPE tissues and will offer new perspectives in studying gene expression in Candida species.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Fulltext Real-time PCR detection of male-specific coliphages

Yong SF, Ngeow YF, Tong YK, Ong JT

Malays J Pathol, 2006 Dec;28(2):79-82.
PMID: 18376795 MyJurnal

Male-specific coliphages are often used as indicators of contamination by enteric viruses. These phages can be detected in water samples by plaque assays and by polymerase chain reaction. In this study, the M13 coliphage was used to develop a real-time PCR assay for the detection of male-specific DNA coliphages. The real-time PCR was found to have a reaction efficiency of 1.45 and detection limit of 10(-3) plaque forming units per reaction mix. Repeated amplification and melting curve analyses demonstrated high specificity and reproducibility of the real-time assay. Quantitative detection with the real-time PCR should allow rapid assessment of the level of viral contamination in water.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction*
Fulltext Potentiality of a triple microRNA classifier: miR-193a-3p, miR-23a and miR-338-5p for early detection of colorectal cancer

Yong FL, Law CW, Wang CW

BMC Cancer, 2013 Jun 08;13:280.
PMID: 23758639 DOI: 10.1186/1471-2407-13-280

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNA molecules that act as regulators of gene expression. Circulating blood miRNAs offer great potential as cancer biomarkers. The objective of this study was to correlate the differential expression of miRNAs in tissue and blood in the identification of biomarkers for early detection of colorectal cancer (CRC).
METHODS: The study was divided into two phases: (I) Marker discovery by miRNA microarray using paired cancer tissues (n = 30) and blood samples (CRC, n = 42; control, n = 18). (II) Marker validation by stem-loop reverse transcription real time PCR using an independent set of paired cancer tissues (n = 30) and blood samples (CRC, n = 70; control, n = 32). Correlation analysis was determined by Pearson's test. Logistic regression and receiver operating characteristics curve analyses were applied to obtain diagnostic utility of the miRNAs.
RESULTS: Seven miRNAs (miR-150, miR-193a-3p, miR-23a, miR-23b, miR-338-5p, miR-342-3p and miR-483-3p) have been found to be differentially expressed in both tissue and blood samples. Significant positive correlations were observed in the tissue and blood levels of miR-193a-3p, miR-23a and miR-338-5p. Moreover, increased expressions of these miRNAs were detected in the more advanced stages. MiR-193a-3p, miR-23a and miR-338-5p were demonstrated as a classifier for CRC detection, yielding a receiver operating characteristic curve area of 0.887 (80.0% sensitivity, 84.4% specificity and 83.3% accuracy).
CONCLUSION: Dysregulations in circulating blood miRNAs are reflective of those in colorectal tissues. The triple miRNA classifier of miR-193a-3p, miR-23a and miR-338-5p appears to be a potential blood biomarker for early detection of CRC.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext A case-controlled validation study of a blood-based seven-gene biomarker panel for colorectal cancer in Malaysia

Yip KT, Das PK, Suria D, Lim CR, Ng GH, Liew CC

J Exp Clin Cancer Res, 2010;29:128.
PMID: 20846378 DOI: 10.1186/1756-9966-29-128

BACKGROUND: Colorectal cancer (CRC) screening is key to CRC prevention and mortality reduction, but patient compliance with CRC screening is low. We previously reported a blood-based test for CRC that utilizes a seven-gene panel of biomarkers. The test is currently utilized clinically in North America for CRC risk stratification in the average-risk North American population in order to improve screening compliance and to enhance clinical decision making.
METHODS: In this study, conducted in Malaysia, we evaluated the seven-gene biomarker panel validated in a North American population using blood samples collected from local patients. The panel employs quantitative RT-PCR (qRT-PCR) to analyze gene expression of the seven biomarkers (ANXA3, CLEC4D, TNFAIP6, LMNB1, PRRG4, VNN1 and IL2RB) that are differentially expressed in CRC patients as compared with controls. Blood samples from 210 patients (99 CRC and 111 controls) were collected, and total blood RNA was isolated and subjected to quantitative RT-PCR and data analysis.
RESULTS: The logistic regression analysis of seven-gene panel has an area under the curve (AUC) of 0.76 (95% confidence interval: 0.70 to 0.82), 77% specificity, 61% sensitivity and 70% accuracy, comparable to the data obtained from the North American investigation of the same biomarker panel.
CONCLUSIONS: Our results independently confirm the results of the study conducted in North America and demonstrate the ability of the seven biomarker panel to discriminate CRC from controls in blood samples drawn from a Malaysian population.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Gene isolation using degenerate primers targeting protein motif: A laboratory exercise

Yeo BPH, Foong LC, Tam SM, Lee V, Hwang SS

Biochem Mol Biol Educ, 2018 01;46(1):47-53.
PMID: 29131478 DOI: 10.1002/bmb.21089

Structures and functions of protein motifs are widely included in many biology-based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding site-leucine rich repeats (NBS-LRR) proteins, successfully used for the isolation and characterization of many plant resistance gene analogues (RGAs), is featured in the development of a series of laboratory experiments using important molecular biology techniques. A set of previously isolated RGA sequences is used as the model for performing sequence alignment and visualising 3D protein structure using current bioinformatics programs (Clustal Omega and Argusdock software). A pair of established degenerate primer sequences is provided for the prediction of targeted amino acids sequences in the RGAs. Reverse transcription-polymerase chain reaction (RT-PCR) is used to amplify RGAs from total RNA samples extracted from the tropical wild relative of black pepper, Piper colubrinum (Piperaceae). This laboratory exercise enables students to correlate specific DNA sequences with respective amino acid codes and the interaction between conserved motifs of resistance genes with putatively targeted proteins. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):47-53, 2018.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction

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