scholarly journals A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

2021 ◽  
Author(s):  
Ridim D Mote ◽  
Shinde Laxmikant V ◽  
Surya Bansi Singh ◽  
Mahak Tiwari ◽  
Hemant Singh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developing Countries ◽  
Real Time ◽  
Real Time Pcr ◽  
Research Funding ◽  
Cost Effective ◽  
Small Enterprises ◽  
Cdna Synthesis ◽  
Efficient Approach ◽  
Cost Effective Approach

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

scholarly journals Quantification of mRNA in Whole Blood by Assessing Recovery of RNA and Efficiency of cDNA Synthesis

2006 ◽  
Vol 52 (4) ◽  
pp. 634-642 ◽  
Author(s):  
Masato Mitsuhashi ◽  
Shigeru Tomozawa ◽  
Katsuya Endo ◽  
Atsushi Shinagawa
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Ex Vivo ◽  
Statistical Significance ◽  
Absolute Quantification ◽  
External Control ◽  
Cdna Synthesis ◽  
Optimized Conditions

Abstract Background: Current gene expression analysis relies on the assumption that the isolated RNA represents all species of mRNA in proportions equal to those in the original materials. No system is available for absolute quantification of mRNA. Methods: We applied whole blood to 96-well filterplates to trap leukocytes. Lysis buffer containing cocktails of specific reverse primers and known concentrations of synthetic external control RNA (RNA34) was added to filterplates, and cell lysates were transferred to oligo(dT)-immobilized microplates for hybridization. We then synthesized the cDNA in the oligo(dT)-immobilized microplates from these primer sites and used the cDNA for real-time PCR. RNA34 acted as a universal control, and gene amplification results were converted to quantities of mRNA per microliter of whole blood after the recovery of RNA34 in each sample was determined. Results: Under fully optimized conditions, both added RNA34 and native mRNA species exhibited ∼10% recovery from whole blood to real-time PCR. When whole blood was stimulated ex vivo, changes in gene expression as low as 30%–40% were detected with statistical significance, and the experimental CVs were low (10%–20%). Conclusion: This new system to estimate mRNA copies per microliter of whole blood may allow standardization of gene-expression–based molecular diagnostics.

scholarly journals Correction to: A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

2022 ◽  
Vol 47 (1) ◽  
Author(s):  
Ridim D Mote ◽  
Shinde Laxmikant V ◽  
Surya Bansi Singh ◽  
Mahak Tiwari ◽  
Hemant Singh ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Efficient Approach

scholarly journals A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

2021 ◽  
Vol 46 (4) ◽  
Author(s):  
Ridim D Mote ◽  
V Shinde Laxmikant ◽  
Surya Bansi Singh ◽  
Mahak Tiwari ◽  
Hemant Singh ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Efficient Approach

Reliable and cost-effective approach for diagnosis of heterozygousF8/F9large deletions by quantitative real-time PCR

Haemophilia ◽  
2015 ◽  
Vol 21 (3) ◽  
pp. e247-e251
Author(s):  
M. M. Abelleyro ◽  
C. P. Radic ◽  
T. Tetzlaff ◽  
V. Marchione ◽  
A. F. Fundia ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Quantitative Real Time Pcr ◽  
Cost Effective Approach

scholarly journals Developing high performance and cost effective real time (PCR) for P-glycoprotein gene expression analysis in rat

2012 ◽  
Vol 11 (52) ◽  
pp. 11501-11508
Author(s):  
Ghasemian Sepideh ◽  
Najafzadeh Hossein ◽  
Reza Seifi Masoud ◽  
Jalali Amir ◽  
Galehdari Hamid
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Analysis ◽  
Real Time Pcr ◽  
High Performance ◽  
Gene Expression Analysis ◽  
Cost Effective ◽  
Glycoprotein Gene ◽  
P Glycoprotein

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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