scholarly journals Disain Primer Aktin Spesifik Tuntun Angin (Elaeocarpus floribundus) (Actin Primer Design Specific of Tuntun Angin (Elaeocarpus floribundus))

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Dewi Indriyani Roslim ◽  
Herman Herman
Keyword(s):  
Internal Control ◽  
Gene Expression Analysis ◽  
Dna Isolation ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Actin Gene ◽  
Chain Reaction ◽  
Reverse Primer ◽  
Dna Fragment ◽  
Polymerase Chain

Abstrak Aktin merupakan salah satu dari housekeeping gene yang dapat digunakan sebagai kontrol internal pada analisis ekspresi gen.  Sampai saat ini, sekuen DNA dari gen penyandi aktin pada tanaman tuntun angin (Elaeocarpus floribundus) belum pernah dipublikasikan. Penelitian ini bertujuan merancang primer aktin spesifik untuk tanaman Elaeocarpus floribundus bedasarkan primer aktin degenerate.  Pasangan primer degenerate rancangan yang digunakan adalah PlAc46S sebagai primer forward: 5’- ATGGTNGGNATGGGNCARAA-3’ dan PlAc245N sebagai primer reverse: 3’- GTDATNACYTGNCCRTCNGG-5’.  Metode penelitian meliputi isolasi DNA dari daun segar, amplifikasi dengan teknik polymerase chain reaction (PCR), elektroforesis pada 1,2% gel agarose, perunutan nukleotida, dan analisis data menggunakan program BLASTn dan perangkat lunak MEGA versi 6.06.  Fragmen DNA aktin telah berhasil diamplifikasi menggunakan primer aktin degenerate dan telah dirancang primer aktin spesifik untuk Elaeocarpus floribundus dengan urutan sebagai berikut:5’-CCAAATCATGTTTGAGACCT-3’(forward) dan 5’-GAACACGTTAATTCCTGCTC-3’ (reverse). Primer tersebut selanjutnya dapat digunakan untuk mengamplifikasi gen aktin dari E. floribundus pada analisis ekspresi gen.Kata Kunci: aktin, Elaeocarpus floribundus, PCR, primer degenerate, tuntun angin. Abstract Actin is one of the housekeeping genes which commonly be used as an internal control in gene expression analysis.  Until now, the DNA sequence of actin gene of tuntun angin (Elaeocarpus floribundus) has never been published. This study aimed to design the actin primer specific for E. floribundus based on degenerate actin primer.  The pair of the degenerate actin primers (PlAc46S 5’- ATGGTNGGNATGGGNCARAA-3’ and PlAc245N 3’- GTDATNACYTGNCCRTCNGG-5’) were used in this study.  Methods included DNA isolation from fresh leaf, PCR, electrophoresis, sequencing, and data analysis using BLASTn program and MEGA software version 6.06.  The actin DNA fragment has been successfully amplified using the degenerate actin primer and then the actin primer specific for E. floribundus with the following sequences: 5’-CCAAATCATGTTTGAGACCT-3’ (forward) and 5’- AACACGTTAATTCCTGCTC-3’ (reverse) have been designed.  The primer pairs can be used to amplify the actin gene of E. floribundus.Keywords: actin, degenerate primer, Elaeocarpus floribundus, PCR, tuntun angin.

scholarly journals Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

2021 ◽  
Author(s):  
Rajeev Kumar Jain ◽  
Nagaraj Perumal ◽  
Rakesh Shrivastava ◽  
Kamlesh Kumar Ahirwar ◽  
Jaya Lalwani ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Specific Gene ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Gene Target ◽  
Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

scholarly journals Transcriptional expression study in the central nervous system of rats: what gene should be used as internal control?

2014 ◽  
Vol 12 (3) ◽  
pp. 336-341 ◽  
Author(s):  
Ana Carolina de Moura ◽  
Virgínia Meneghini Lazzari ◽  
Grasiela Agnes ◽  
Silvana Almeida ◽  
Márcia Giovenardi ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Internal Control Gene ◽  
Chain Reaction ◽  
Beta Actin ◽  
The Central Nervous System ◽  
Polymerase Chain ◽  
The Brain

Objective A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex). Methods Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder. Results Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively. Conclusion Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene.

scholarly journals Isolation of Housekeeping Genes on Durik-durik (Syzygium sp)

2018 ◽  
Vol 10 (2) ◽  
pp. 237-244
Author(s):  
Dewi Indriyani Roslim ◽  
Liza Aulia Yusfi ◽  
Desriani Ritawati Hutagalung ◽  
Niswah Faizah ◽  
Lestari Andriani ◽  
...  
Keyword(s):  
Internal Control ◽  
18S Rrna ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Internal Controls ◽  
Expression Data ◽  
Bioinformatic Tools ◽  
Normalize Gene Expression ◽  
Total Dna ◽  
Polymerase Chain

Housekeeping gene is a gene expressed with a fixed level and in abundant amounts under various conditions. After validation, the housekeeping gene can be used as an internal control to normalize gene expression data. This study reports the isolation of several housekeeping genes in Durik-durik plant (Syzygium sp). This plant material in form of fresh leaves from Durik-durik plants are taken from Kajuik Lake, Riau Province. The next stage is total DNA isolation, polymerase chain reaction, electrophoresis, sequencing and data analysis using bioinformatic tools. The isolated housekeeping genes included 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Beta-tubulin and Ubiquitin with sizes of 400 bp, 679 bp, 1134 bp, 836 bp, 1167 bp and 2155 bp, respectively. In addition to 18S rRNA, the five housekeeping genes are the first reported from the genus Syzygium and referable to isolate housekeeping genes in other species in this genus. The six housekeeping genes can be used as internal controls on Durik-durik plants after validation.

Evaluation of polymerase chain reaction and DNA isolation protocols for detection of genetically modified soybean

2007 ◽  
Vol 42 (10) ◽  
pp. 1249-1255 ◽  
Author(s):  
Cibele dos Santos Ferrari ◽  
Luciana Lehmkuhl Valente ◽  
Fábio Cristiano Angonesi Brod ◽  
Caroline Tagliari ◽  
Ernani Sebastião Sant'Anna ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Isolation ◽  
Genetically Modified ◽  
Chain Reaction ◽  
Genetically Modified Soybean ◽  
Polymerase Chain

DNA Isolation from Recalcitrant Materials Such as Tree Roots, Bark, and Forest Soil for the Detection of Fungal Pathogens by Polymerase Chain Reaction

1998 ◽  
Vol 262 (1) ◽  
pp. 79-82 ◽  
Author(s):  
Günther Bahnweg ◽  
Steffen Schulze ◽  
Evelyn M. Möller ◽  
Hilkea Rosenbrock ◽  
Christian Langebartels ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Forest Soil ◽  
Fungal Pathogens ◽  
Dna Isolation ◽  
Chain Reaction ◽  
Tree Roots ◽  
Polymerase Chain

scholarly journals A PCR-based Assay for Distinguishing between A1 and A2 Mating Types of Phytophthora capsici

2017 ◽  
Vol 142 (4) ◽  
pp. 260-264
Author(s):  
Ping Li ◽  
Dong Liu ◽  
Min Guo ◽  
Yuemin Pan ◽  
Fangxin Chen ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Mating Type ◽  
Phytophthora Capsici ◽  
Mating Types ◽  
Sequence Repeat ◽  
Chain Reaction ◽  
Plant Parasite ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Microsatellite Diversity

Sexual reproduction in the plant parasite Phytophthora capsici Leonian requires the interaction of two distinct mating types, A1 and A2. Co-occurrence of these mating types can enhance the genetic diversity of P. capsici and alter its virulence or resistance characteristics. Using an intersimple sequence repeat (ISSR) screen of microsatellite diversity, we identified, cloned, and sequenced a novel 1121-base pair (bp) fragment specific to the A1 mating type of P. capsici. Primers Pcap-1 and Pcap-2 were designed from this DNA fragment to specifically detect the A1 mating type. Polymerase chain reaction (PCR) using these primers amplified an expected 997-bp fragment from known A1 mating types, but yielded a 508-bp fragment from known A2 mating types. This PCR-based assay could be adapted to accurately and rapidly detect the co-occurrence of A1 and A2 P. capsici mating types from field material.

scholarly journals Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

1996 ◽  
Vol 44 (10) ◽  
pp. 1205-1207 ◽  
Author(s):  
A Dakhama ◽  
V Macek ◽  
J C Hogg ◽  
R G Hegele
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Housekeeping Gene ◽  
Chain Reaction ◽  
Viral Genomes ◽  
Negative Results ◽  
Beta Actin ◽  
Target Nucleic Acid ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

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