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

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


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 



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.


  1. 1.
    Gibson UE, Heid CA, Williams PM (1996) A novel method for real time quantitative RT-PCR. Genome Res 6:995–1001CrossRefGoogle Scholar
  2. 2.
    Higuchi R, Dollinger G, Walsh PS, Griffith R (1992) Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10:413–417CrossRefGoogle Scholar
  3. 3.
    Bustin SA, Mueller R (2005) Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin Sci (Lond) 109:365–379CrossRefGoogle Scholar
  4. 4.
    Bustin SA, Nolan T (2004) Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 15:155–166PubMedPubMedCentralGoogle Scholar
  5. 5.
    Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39CrossRefGoogle Scholar
  6. 6.
    Radstrom P, Knutsson R, Wolffs P, Lovenklev M, Lofstrom C (2004) Pre-PCR processing: strategies to generate PCR-compatible samples. Mol Biotechnol 26:133–146CrossRefGoogle Scholar
  7. 7.
    Wolffs P, Grage H, Hagberg O, Radstrom P (2004) Impact of DNA polymerases and their buffer systems on quantitative real-time PCR. J Clin Microbiol 42:408–411CrossRefGoogle Scholar
  8. 8.
    Hoorfar J, Wolffs P, Radstrom P (2004) Diagnostic PCR: validation and sample preparation are two sides of the same coin. APMIS 112:808–814CrossRefGoogle Scholar
  9. 9.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25:402–408CrossRefGoogle Scholar

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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