scholarly journals Quantitative RT-PCR using the TaqMan® Gene Expression Assays on StepOnePlus™ Real-Time PCR System v1 (protocols.io.2v8ge9w)

protocols.io ◽  
2019 ◽  
Author(s):  
Lisa Maria ◽  
Giang Tong ◽  
Katharina Schmitt
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Rt Pcr

scholarly journals Beta-2 microglobulin a robust reference housekeeping genes for RNA expression normalization in real time PCR on human leukocytes

2019 ◽  
Author(s):  
Clara MILHEM ◽  
Céline INGELAERE ◽  
Olivier MORALES ◽  
Nadira DELHEM
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Housekeeping Genes ◽  
Internal Controls ◽  
Rna Expression ◽  
Rt Pcr ◽  
Genomic Study ◽  
Beta 2 ◽  
Beta 2 Microglobulin

Abstract Background: Changes in gene expression are increasingly used to evaluate the effects of exposure to environmental agents. Housekeeping genes (Hk) are essential in these analyzes as internal controls to normalize expression levels assessed in real-time PCR (RT-PCR). Ideal Hk genes (i) are constitutively expressed; (ii) do not respond to external stimuli and (iii) show small or no variation between samples or from one assay to another. Previous studies indicate that some commonly used Hk genes, including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, have differential expression in various cell lines. The objective of this study is to identify and validate the most appropriate housekeeping genes for RNA expression analysis of human primary peripheral blood mononuclear cells (PBMCs) in response to antigen like stimulation. Methods: The expression of the five most commonly used genes for a genomic study of lymphocytes was determined [HPRT, 18S, GAPDH, Ubiquitin C (UBC), beta-2 microglobulin (B2M)] on PBMCs, isolated from fresh blood, following activation with anti-CD3 and anti-CD28. This leads to the activation and proliferation of T lymphocytes in order to modulate the expression of specific genes. Results : Using reverse transcriptase RT-PCR protocol, we show that following activation only B2M remain unchanged in PBMCs. This result has been confirmes In contrast, 18S, HPRT and GAPDH show significant variation in PBMC gene expression. Conclusion: Although our results suggest that the relevance of Hk genes should be determined for each experimental condition, B2M appear to be excellent candidate as internal controls. Keywords : Housekeeping genes, RT-PCR, Human primary PBMC.

SPAN-Xb Expression in Myeloma Cells Can Be Upregulated by Pharmacologics.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4837-4837
Author(s):  
Zhiqing Wang ◽  
Yana Zhang ◽  
Jian Zhang ◽  
Seah H. Lim
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tumor Vaccine ◽  
Rt Pcr ◽  
Gm Csf ◽  
Copy Numbers ◽  
Ct Antigen ◽  
Rpmi 8226

Abstract SPAN-Xb is a spermatid-specific protein. We recently identified SPAN-Xb as a novel Cancer-Testis (CT) antigen in multiple myeloma (MM) (Blood2003; 101: 955–960) and that it frequently elicited immune responses in vivo in these patients, suggesting that SPAN-Xb may be a suitable target for immunotherapy of MM. SPAN-Xb, however, was only detected in the tumor cells from 20% of MM patients. To increase the applicability of a SPAN-Xb tumor vaccine for MM, we have set out to determine if SPAN-Xb expression could be upregulated by pharmacologics in 8 MM cell lines. Since most CT antigen expression occurs due to promoter demethylation, we first determined the ability of a hypomethylating agent, 5-azacytidine, to upregulate SPAN-Xb expression. Prior to exposure to 5-azacytidine, SPAN-Xb transcripts were only detected by RT-PCR in ARP-1 and RPMI 8226 cells. Following incubation in 5-azacytidine (2 uM) for 96 hours, the levels of SPAN-Xb transcripts were upregulated, as determined by RT-PCR and real time PCR, in all MM cell lines (ARK-B, ARP-1, H929, IM9, MM1-R, RPMI 8226 and U266) except KMS-11 (Table 1). This was associated with a corresponding increase in the level of SPAN-Xb protein expression, as shown by immunocytochemistry using a murine monoclonal antibody directed at SPAN-Xb. We next determined if SPAN-Xb gene expression could be increased by cytokine pre-incubation of these cells. IL-2 (100 IU/ml) and IL-4 (1000 IU/ml) did not affect SPAN-Xb gene expression. On their own, IL-7 (0.1 ng/ml) and GM-CSF (10 U/ml) did not affect SPAN-Xb gene expression significantly. However, when combined, especially with 5-azacytidine, a summative effect of these pharmacologics on SPAN-Xb gene expression, as determined by real time PCR, was observed in IM9 cells (Table 2). Our results indicate the potential to upregulate SPAN-Xb expression in MM for therapeutic purpose using 5-azacytidine, GM-CSF and IL-7 and provide insight into the possible mechanisms of regulation of SPAN-Xb gene expression. Table 1 Cells pre-5-azacytidine post-5-azacytidine SPAN-Xb mRNA copy numbers before and after 5-azacytidine exposure ARK-B 0 456 ARP-1 260 1376 H929 0 116 IM9 0 532 KMS-11 0 0 MM1-R 0 428 RPMI 8226 272 1348 U266 0 984 Table 2 Pharmacologics SPAN-Xb mRNA SPAN-Xb mRNA copy numbers after exposure to combination pharmacologics 5-azacytidine 532 IL-7 4 GM-CSF 9 IL-7 + GM-CSF 36 5-azacytidine + IL-7 1088 5-azacytidine + GM-CSF 952 5-azacytidine + IL-7 + GM-CSF 1632 medium only 0

scholarly journals Gel express: a novel frugal method quantifies gene relative expression in conventional RT-PCR

2022 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohamed Hazman
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Express Method ◽  
Relative Expression ◽  
Crossing Point

Abstract Background Real-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression. Nevertheless, compared with conventional PCR, the real-time PCR system is extremely expensive and not affordable for limited or mid-budget research laboratories. Here, a novel, doable and low-cost recipe (referred to as gel express) is developed to quantify gene expression using conventional RT-PCR assay. The novelty of the gel express method is based on replacing crossing point (CP) values with integrated density (IntDen) values of PCR amplicon bands in real-time PCR regular mathematical formulas. Results In this work, gene expression profiles of two different rice stress-marker genes (OsCYP94C2a and OsLOX8) were quantified in response to mechanical wounding at different time points (0, 30, 60, and 150 min). In the gel express method, the free software ImageJ was employed to measure integrated density (IntDen) values of PCR amplicon bands in agarose gel images. IntDen values were then used instead of crossing point (CP) values according to the following modified formula: [EIntDen(ref)/EIntDen(target)]sample ÷ [EIntDen(ref)/EIntDen(target)]control. Gene relative expression profiles (dynamic expression pattern) quantified by gel express method in both genes were highly comparable with real-time RT-PCR. R2 values were 0.9976 and 0.9975 in OsCYP94C2a and OsLOX, respectively. PCR amplification efficiency (E) for all studied genes could be calculated depending on IntDen values through experimentally designed calibration curves. PCR amplification efficiencies with all studied genes obtained by gel express were all in the accepted range. For better-visualized PCR amplicons thus detectable biological effects between treatments, the number of PCR cycles applied in gel express method (IntCyc) was experimentally estimated to be 29 cycles. Conclusions Gel express is a novel, cost-effective and feasible recipe for quantifying gene relative expression in conventional RT-PCR. The expression pattern quantified by gel express is highly comparable and fits the expression data revealed by the used real-time PCR system.

scholarly journals Pengaruh Variasi Jumlah Tembakan Nanosecond Pulsed Electric Fields (Nspefs) Terhadap Ekspresi Gen Socs3 pada Sel Kanker Serviks Hela S3

2017 ◽  
Vol 1 (2) ◽  
pp. 45
Author(s):  
Martina Kurnia Rohmah
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Electric Fields ◽  
Real Time Pcr ◽  
Pulsed Electric Fields ◽  
Rt Pcr ◽  
Kolmogorov Smirnov ◽  
Hela S3 ◽  
Short Time ◽  
Research Show

AbstrakNanosecond Pulsed Electric Fields (NsPEFs) merupakan teknologi bioelektrik yang berkembang dari teknologi elektroporasi. NsPEFs diberikan dengan intensitas tinggi namun dalam waktu yang sangat singkat yaitu 1 - 300 nanosekon. NsPEFs terbukti memiliki sejumlah efek biologis dan telah banyak dikembangkan dalam berbagai terapi salah satunya pada terapi kanker. Pada kanker serviks, protein HPV dapat menekan sejumlah ekspresi supresor tumor salah satunya yaitu gen Socs3. Penelitian ini bertujuan untuk mengetahui pengaruh perbedaan jumlah tembakan NsPEFs pada ekspresi gen Socs3. Sel HeLa S3 dikultur pada medium α-MEM dengan serum FBS 10%. Sebesar 20 kV/cm dalam durasi 80 ns NsPEFs dipapar pada suspensi sel dalam 4 mm cuvette. Gelombang NsPEFs dideteksi oleh probe bervoltase tinggi pada Oscilloscope. NsPEFs diberikan pada 0, 5, 10, 20, 30, 40, 0, dan 60 kali tembakan. Analisis ekspresi gen dilakukan dengan dua metode yaitu kuantitatif menggunakan Real time PCR dan kualitatif dengan RT-PCR. Data kuantitatif dianalisis secara statistik menggunakan Kolmogorf-Smirnov, Anova dan HSD Tukey (p<0.05). Hasil studi ini membuktikan bahwa paparan NsPEFs berpengaruh secara signifikan pada ekspresi gen Socs3 (p=0.000). Jumlah tembakan optimal 20 dan 30 kali dapat meningkatkan ekspresi gen Socs3 berturut-turut sebanyak = 2.779 dan = 3.105 kali. Ekspresi gen Socs3 akan menurun pada tembakan di atas 30 tembakan. Kata Kunci: NsPEFs, tembakan, ekspresi, Socs3 AbstractNanosecond Pulsed Electric Fields (NsPEFs) is bioelectric that was developed by electroporation technology. NsPEFs use high intensity in short time exposure (1 – 300 nanosecond). NsPEFs have biological effect and was developed in cancer therapy. In cervical cancer, viral protein of HPV depresses some tumor suppressors like Socs3 gene. This research aims to investigate the effect of short variation in Socs3 gene expression. HeLa S3 cells were cultured in α-MEM with FBS 10%. NsPEFs as much as 20 kV/cm and 80 nano seconds was exposure over HeLa S3 cell in 4 mm cuvette. Wave of NsPEFs was detected by high voltage probe in oscilloscope. NsPEFs was exposure at 0 (control), 5, 10, 20, 30, 40, 50, and 60 shots. Socs3 gene expression was analyzed using real time PCR and RT-PCR. Quantitative data was analyzed by Kolmogorov-Smirnov, Anova, and HSD Tuker (p<0.05). This research show that NsPEFs is significantly increase Socs3 gene expression (p=0.000). The optimal shot 20 and 30 shots increase Socs3 gene expression subsequently = 2.779 and = 3.105 times. This expression decrease in higher than 30 shots of NsPEFs exposure. Keywords: NsPEFs, shot, expression, Socs3

Respon Morfologis dan Ekspresi Gen Aquaporin pada Padi IR 64 yang Mengalami Cekaman Kekeringan pada Fase Reproduktif (Morphological Responses and Aquaporin Gene Expression in Rice IR64 under Drought Stress at the Reproductive Stage)

2018 ◽  
Vol 7 (2) ◽  
Author(s):  
Made Pharmawati ◽  
Ni Nyoman Wirasiti ◽  
Luh Putu Wrasiati
Keyword(s):  
Gene Expression ◽  
Drought Stress ◽  
Real Time ◽  
Real Time Pcr ◽  
Control Plant ◽  
Reproductive Stage ◽  
Limiting Factors ◽  
Rice Grain ◽  
Aquaporin Gene ◽  
Peg Treatment

Abstrak Cekaman kekeringan merupakan faktor pembatas penting bagi pertumbuhan dan produktivitas tanaman termasuk padi.      Penelitian ini bertujuan menganalisis respon padi IR64 terhadap cekaman kekeringan dengan pemberian polietilen glikol (PEG) pada fase reproduktif.  Penelitian juga bertujuan menganalisis ekspresi gen aquaporin akibat cekaman kekeringan.  Bibit padi ditanam dalam pot dan perlakuan PEG dengan konsentrasi 108g/L (-0.25MPa) dan 178g/L (-0.52 MPa) diberikan saat munculnya panikula. Perlakuan diberikan selama 2 minggu, kemudian tanaman disiram kembali.  Ekspresi gen diamati pada akhir perlakuan dengan semi kuantitatif real time PCR.  Ekstraksi RNA menggunakan RNeasy plant mini kit, sedangkan sintesis cDNA menggunakan Transcriptor First Strand cDNA Kit.  Hasil penelitian menunjukkan bahwa jumlah malai dan berat total malai berkurang akibat cekaman kekeringan.  Persentase gabah kosong mencapai 84,6% pada perlakuan PEG-0,52 MPa, sedangkan pada perlakuan PEG -0,25 MPa persentase gabah kosong sebesar 67,8%.  Pada kontrol persentase gabah kosong adalah 10,3%.  Ekspresi gen OsPIP2;7 sedikit menurun pada perlakuan PEG -0,52 MPa.Kata kunci: ekspresi gen, IR64, kekeringan, padi, PEG  Abstract Drought stress is one of the limiting factors of plant growth and productivity including rice.  The aim of this study was to analyze responses of IR64 rice to polyethylene glycol (PEG)-induced-drought stress at the reproductive stage.  This study also aimed to analyze the expression of aquaporin under drought stress.  Rice seedlings were grown in pot system and PEG treatment at concentration of -0.25MPa (108g/L) and -0.52 MPa (178g/L) were given when the panicles arose.  Treatments were conducted for 2 weeks, after that the plants were rewatered.  Gene expression was evaluated at the end of PEG treatment using semi quantitative real time PCR. RNA was extracted using RNeasy plant mini kit, while cDNA synthesis was done using Transcriptor First Strand cDNA Kit.  The results showed that the number and weight of rice ear were less in plant treated with PEG than in control.  The percentage of empty rice grain reached 84.6% at PEG -0.52 MPa, while at PEG -0.25 MPa the percentage of empty grain was 67.8%.  In control plant, the percentage of empty grain was 10.3%.  Drought stress did not alter the expression of OsPIP2;7.  Keywords: drought, gene expression, IR64, PEG, rice

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