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Quantitative Real-Time PCR for Evaluating Transcriptional Changes in T-Lymphocytes

  • Atish KizhakeyilEmail author
  • Navin Kumar Verma
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1930)

Abstract

The real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an indispensable technology that enables reliable transcriptional analysis routinely used in molecular biology studies. The qRT-PCR technique quantifies mRNA by taking advantage of the reverse transcriptase-dependent conversion of RNA into cDNA and subsequent amplification of the cDNA using PCR. The amount of PCR product is directly proportional to the initial starting quantity of mRNA. The straightforward but complex methodologies used in this technique involve multiple sequential steps that include isolation of mRNA, conversion of mRNA into cDNA, amplification of the cDNA, and quantification of amplicons. In this chapter, we describe an optimized protocol for performing qRT-PCR in human T-lymphocytes.

Key words

qRT-PCR Gene expression analysis mRNA quantification Reverse transcriptase 

Notes

Acknowledgments

This work was supported in part by grants from Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant to N.K.V. and the Singapore Ministry of Education under its Singapore Ministry of Education (MOE) Academic Research Fund (AcRF) Tier 2 Grant (MOE2017-T2-2-004). A.K. acknowledges Ph.D. fellowship provided by Lee Kong Chian School of Medicine, Nanyang Technological University Singapore.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of MedicineNanyang Technological University SingaporeSingaporeSingapore
  2. 2.Lee Kong Chian School of MedicineNanyang Technological University SingaporeSingaporeSingapore

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