PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency

Suslov, O.

doi:10.1093/nar/gni176

Cited by 121Molecular Biology Techniques and ApplicationsRNA Research and SplicingRNA and protein synthesis mechanisms

PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency

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Suslov, O.·2005·Nucleic Acids Research

DOI 10.1093/nar/gni176PMC PMC1298932

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Abstract

This study addresses the problem of PCR inhibition by reverse transcriptase. It has been shown that the inhibition occurs mostly when a small amount of RNA is taken for RT reaction, and it is more visible for rarely expressed transcripts. We show here that the inhibition takes place regardless of what amount of template is utilized for RT. The inhibition possesses a global nature, i.e. the amplification of any given transcript may be compromised with different levels of inhibition. The process of inhibition also explains wrongfully derived PCR amplification efficiencies, sometimes more than 100%, when the sequential dilutions of unpurified RT sample are utilized to build the calibration curve. The RT influences PCR not only by inhibiting it. When microgram(s) of RNA are taken for RT reaction, reverse transcriptase may cause overamplification of some transcripts under certain PCR conditions. The possible mechanism of RT influence on PCR is presented, and a purification method is implemented to remove the effects of RT on PCR.

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The root cause of poor quality qPCR data is typically inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing.
“The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing.”
From Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data (2017) · cites this paper as [18]
The Discussion (page unavailable) says residual reverse transcriptase can inhibit PCR and that dilution reduces this inhibition, supporting the concern about insufficient dilution of residual protein. It does not establish that this is typically the root cause of poor-quality qPCR data, or that chemical contaminants variably inhibit primer annealing. (LLM, conf=0.86)
Experimental — accuracy not yet measured
In qPCR, components of the reverse transcription reaction can act as inhibitors of Taq polymerase, altering reaction efficiency and Cq values to give artifactual qPCR data if not adequately diluted.
“However, in the case of miRNA and mRNA, the components of the reverse transcription (RT) reaction can also act as inhibitors of Taq polymerase, altering reaction efficiency and the associated Cq values to give artifactual qPCR data if not adequately diluted (Fig. 2)18.”
From Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data (2017) · cites this paper as [18]
The Introduction (page not available) says RT reaction components inhibit PCR and that extensive dilution is intended to minimize the effect. The Results (page not available) report altered Ct values in unpurified RT samples, and the Discussion links inhibition to misleading amplification efficiencies. The paper examines mRNA transcripts but does not establish the claim specifically for miRNA assays. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Subtle differences in the level of impurities between samples even under rigorous RNA extraction can alter Taq activity, imparting gross variability on resulting data.
“However, there are frequently subtle differences in the level of impurities between samples even under highly rigorous RNA extraction procedures that can alter Taq activity, imparting gross variability on the resulting data18, 19.”
From Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data (2017) · cites this paper as [18]
The Results and Discussion (page not provided) show that residual reverse transcriptase can interfere with Taq polymerase and that purifying RT reactions reduces variability in real-time PCR. They do not establish that subtle differences in impurities between samples persist despite rigorous RNA extraction or cause gross variability. (LLM, conf=0.86)
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Lv, X., Li, Y., Qiu, W., Wu, X., Xu, B., Liang, Y., Liu, B., Shao-jun, C., Rao, P., & Ni, L. (2016) · Food Control · doi:10.1016/j.foodcont.2016.01.040 · Cited by 45 · see citing works
Variables influencing the efficiency and interpretation of reverse transcription quantitative PCR (RT-qPCR): An empirical study using Bacteriophage MS2
Miranda, J. A., & Steward, G. F. (2016) · Journal of Virological Methods · doi:10.1016/j.jviromet.2016.12.002 · Cited by 43 · see citing works
“There have been many reports of inhibition of qPCR by RT reaction components, whether the RT enzyme itself (Chandler, Wagnon, and Bolton, 1998; Chumakov, 1994; Suslov and Steindler, 2005) or other components of the reaction (Levesque-Sergerie et al., 2007).”
Background
Substantial performance discrepancies among commercially available kits for reverse transcription quantitative polymerase chain reaction: A systematic comparative investigator-driven approach
Sieber, M. W., Recknagel, P., Glaser, F., Witte, O. W., Bauer, M. W., Claus, R. A., & Frahm, C. (2010) · Analytical Biochemistry · doi:10.1016/j.ab.2010.03.007 · Cited by 43 · see citing works
Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP
Khorosheva, E. M., Karymov, M. A., Selck, D. A., & Ismagilov, R. F. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv877 · Cited by 42 · see citing works
“Estimation of HCV RNA concentration using dRT-PCR amplification on the SlipChip device HCV viral RNA was added to the RT-PCR mix, which contained the following: 20 l of 2X SsoFast EvaGreen SuperMix, 1.0 l of each PCR primer (10 M), 2.0 l of BSA solution (20 mg/ml), 1.0 l of SuperScript R© III Reverse Transcriptase, 1.…”
Influential
Suggested guidelines for validation of real-time PCR assays in veterinary diagnostic laboratories
Toohey-Kurth, K. L., Reising, M. M., Tallmadge, R. L., Goodman, L. B., Bai, J., Bolin, S. R., Pedersen, J. C., Bounpheng, M. A., Pogranichniy, R. M., Christopher‐Hennings, J., et al. (2020) · Journal of Veterinary Diagnostic Investigation · doi:10.1177/1040638720960829 · Cited by 43 · see citing works
The Future of Digital Polymerase Chain Reaction in Virology
Vynck, M., Trypsteen, W., Thas, O., Vandekerckhove, L., & Spiegelaere, W. D. (2016) · Molecular Diagnosis & Therapy · doi:10.1007/s40291-016-0224-1 · Cited by 40 · see citing works
Biporous silica nanostructure-induced nanovortex in microfluidics for nucleic acid enrichment, isolation, and PCR-free detection
Jeon, E., Koo, B., Kim, S., Kim, J., Yu, Y., Jang, H., Lee, M., Kim, S., Kang, T., Kim, S. K., et al. (2024) · Nature Communications · doi:10.1038/s41467-024-45467-w · Cited by 39 · see citing works
Tutorial: Guidelines for Single-Cell RT-qPCR
Žucha, D., Kubista, M., & Valihrach, L. (2021) · Cells · doi:10.3390/cells10102607 · Cited by 39 · see citing works
“Here, 10× cDNA dilution is mandatory to prevent PCR inhibition by RT reagents [54,58,83,84].”
Method
Regulation of gene expression in human mammary epithelium: effect of breast pumping
Maningat, P. D., Sen, P., Sunehag, A. L., Hadsell, D. L., & Haymond, M. W. (2007) · Journal of Endocrinology · doi:10.1677/joe-07-0394 · Cited by 39 · see citing works
The effect of continuous and interval exercise on PGC‐1α and PDK4 mRNA in type I and type II fibres of human skeletal muscle
Wang, L., & Sahlin, K. (2011) · Acta Physiologica · doi:10.1111/j.1748-1716.2011.02354.x · Cited by 38 · see citing works
“This is probably related to disturbance of the PCR reaction by some component in the RT buffer (Suslov & Steindler 2005) similar to that observed when establishing the calibration curves of primers.”
PCR inhibitors and facilitators – Their role in forensic DNA analysis
Vajpayee, K., Dash, H. R., Parekh, P. B., & Shukla, R. K. (2023) · Forensic Science International · doi:10.1016/j.forsciint.2023.111773 · Cited by 37 · see citing works
Higher random oligo concentration improves reverse transcription yield of cDNA from bioptic tissues and quantitative RT-PCR reliability
Nardon, E., Donada, M., Bonin, S., Dotti, I., & Stanta, G. (2009) · Experimental and Molecular Pathology · doi:10.1016/j.yexmp.2009.07.005 · Cited by 35 · see citing works
Competitiveness of Quantitative Polymerase Chain Reaction (qPCR) and Droplet Digital Polymerase Chain Reaction (ddPCR) Technologies, with a Particular Focus on Detection of Antibiotic Resistance Genes (ARGs)
Park, S., Rana, A., Sung, W., & Munir, M. (2021) · Applied Microbiology · doi:10.3390/applmicrobiol1030028 · Cited by 32 · see citing works
Quantitative assessment of short amplicons in FFPE-derived long-chain RNA
Kong, H., Zhu, M., Cui, F., Wang, S., Gao, X. J., Lu, S., Wu, Y., & Zhu, H. (2014) · Scientific Reports · doi:10.1038/srep07246 · Cited by 31 · see citing works
V-gene amplification revisited – An optimised procedure for amplification of rearranged human antibody genes of different isotypes
Lim, T. S., Mollova, S., Rubelt, F., Sievert, V., Dübel, S., Lehrach, H. R., & Konthur, Z. (2010) · New Biotechnology · doi:10.1016/j.nbt.2010.01.001 · Cited by 30 · see citing works
“Using high amounts of RNA, the RTase could cause an overamplification of certain transcripts, leading to misrepresentation of individual transcript populations [ 30 ].”
Background
VCP expression decrease as a biomarker of preclinical and early clinical stages of Parkinson’s disease
Alieva, A. K., Rudenok, M. M., Filatova, E. V., Karabanov, A. V., Doronina, O. B., Doronina, K. S., Kolacheva, A. A., Ugrumov, M. V., Illarioshkin, S. N., Slominsky, P. A., et al. (2020) · Scientific Reports · doi:10.1038/s41598-020-57938-3 · Cited by 28 · see citing works
Comparative Expression Analysis of Reference Genes in Field-Grown Cassava
Salcedo, A., Zambrana-Echevarría, C., & Siritunga, D. (2014) · Tropical Plant Biology · doi:10.1007/s12042-014-9137-5 · Cited by 28 · see citing works
Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA
Okello, J. B. A., Rodriguez, L., Poinar, D., Bos, K. I., Livex, O. A., Bimenya, G. S., Sewankambo, N. K., Henry, K. R., Kuch, M., & Poinar, H. N. (2010) · PLoS ONE · doi:10.1371/journal.pone.0013931 · Cited by 28 · see citing works
“copy number RNA levels will depend mainly on the reverse transcription step [8,10,11], it is known that certain reaction components have inhibitory effects on downstream applications, especially qPCR [5,12,13,14,15,16].”
BackgroundInfluential
Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification
Ma, Y., Teng, F., & Libera, M. (2018) · Analytical Chemistry · doi:10.1021/acs.analchem.8b00058 · Cited by 27 · see citing works
Potential authentication of various meat‐based products using simple and efficient DNA extraction method
Mokhtar, N. F. K., Sheikha, A. F. E., Azmi, N. I., & Mustafa, S. (2019) · Journal of the Science of Food and Agriculture · doi:10.1002/jsfa.10183 · Cited by 27 · see citing works
Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR
Vendrame, M., Manzano, M., Comi, G., Bertrand, J., & Iacumin, L. (2014) · Food Microbiology · doi:10.1016/j.fm.2014.03.010 · Cited by 26 · see citing works
Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors
Arezi, B., McCarthy, M., & Hogrefe, H. H. (2010) · Analytical Biochemistry · doi:10.1016/j.ab.2010.01.024 · Cited by 25 · see citing works
Transcriptome Profile Changes in Mice with MPTP-Induced Early Stages of Parkinson’s Disease
Alieva, A. K., Filatova, E. V., Kolacheva, A. A., Rudenok, M. M., Slominsky, P. A., Ugrumov, M. V., & Shadrina, M. I. (2016) · Molecular Neurobiology · doi:10.1007/s12035-016-0190-y · Cited by 25 · see citing works
Mitigation of the Effect of Variability in Digital PCR Assays through use of Duplexed Reference Assays for Normalization
Coulter, S. J. (2018) · BioTechniques · doi:10.2144/btn-2018-0058 · Cited by 25 · see citing works
“Also, since components of the RT reaction have been shown to inhibit subsequent PCR, it might be more susceptible to this type of inhibition because the RT components would not be diluted as in two-step reactions where the mRNA is reverse transcribed separately [26].”
BackgroundMethod
Comparison of reverse transcription–quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis
Fox, B. C., Devonshire, A. S., Baradez, M., Marshall, D., & Foy, C. A. (2012) · Analytical Biochemistry · doi:10.1016/j.ab.2012.05.010 · Cited by 22 · see citing works
“The Cells-to-CT kit was significantly inhibited by the presence of the reverse transcriptase in the qPCR, a wellcharacterized phenomenon observed in previous studies [18,19,34].”
BackgroundMethodResult
Detection and quantitation of Solenopsis invicta virus in fire ants by real-time PCR
Hashimoto, Y., Valles, S. M., & Strong, C. A. (2006) · Journal of Virological Methods · doi:10.1016/j.jviromet.2006.11.008 · Cited by 22 · see citing works
“A number of factors can influence the viral infection value obtained by QPCR, including incomplete nucleic acid purification and recovery, suboptimal reverse transcription or PCR efficiencies, variation in the standards, pipetting/mathematical errors when extrapolating to the organismal level, polymerase inhibition, R…”
Background
Development of RT-qPCR assays for the detection of three latent viruses of pome
Beaver-Kanuya, E., & Harper, S. J. (2020) · Journal of Virological Methods · doi:10.1016/j.jviromet.2020.113836 · Cited by 19 · see citing works
A Single Amino Acid Change to Taq DNA Polymerase Enables Faster PCR, Reverse Transcription and Strand-Displacement
Barnes, W. M., Zhang, Z., & Kermekchiev, M. B. (2021) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2020.553474 · Cited by 19 · see citing works
Evaluation of an Extraction Method for the Detection of GI and GII Noroviruses in Fruit and Vegetable Salads
Cheng, D., Zou, S., Liao, N., Shi, X., Chen, J., Zhang, Y., Sun, L., & Zhang, R. (2017) · Journal of Food Science · doi:10.1111/1750-3841.14022 · Cited by 19 · see citing works
Single molecule transcription profiling with AFM
Reed, J. C., Mishra, B., Pittenger, B., Magonov, S., Troke, J. J., Teitell, M. A., & Gimzewski, J. K. (2006) · Nanotechnology · doi:10.1088/0957-4484/18/4/044032 · Cited by 18 · see citing works
Development of real-time RT-PCR assays for two viruses infecting pome fruit
Beaver-Kanuya, E., Szostek, S. A., & Harper, S. J. (2019) · Journal of Virological Methods · doi:10.1016/j.jviromet.2018.12.008 · Cited by 18 · see citing works
Housekeeping Genes for Parkinson’s Disease in Humans and Mice
Alieva, A. K., Filatova, E. V., Rudenok, M. M., Slominsky, P. A., & Shadrina, M. I. (2021) · Cells · doi:10.3390/cells10092252 · Cited by 17 · see citing works
“Single-stranded DNA was synthesized using 120 ng of total RNA, 100 ng of Escherichia coli tRNA as a carrier [15], specific primers, and the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fischer Scientific Inc.”
Method
Immobilization of microalgae cells in alginate facilitates isolation of DNA and RNA
López, B. R., Hernández, J.-P., Yoav Bashan, A., & de‐Bashan, L. E. (2017) · Journal of Microbiological Methods · doi:10.1016/j.mimet.2017.02.005 · Cited by 17 · see citing works
“One of the reasons for late amplification could be the presence of PCR inhibitors, such as traces of phenol (Suslov and Steindler, 2005).”
Background
Expression analysis of genes involved in mitochondrial biogenesis in mice with MPTP-induced model of Parkinson's disease
Rudenok, M. M., Alieva, A. K., Starovatykh, J. S., Nesterov, M. S., Stanishevskaya, V. A., Kolacheva, A. A., Ugryumov, M. V., Slominsky, P. A., & Shadrina, M. I. (2020) · Molecular Genetics and Metabolism Reports · doi:10.1016/j.ymgmr.2020.100584 · Cited by 16 · see citing works
“Before being added to the reaction mix, cDNA was diluted in an aqueous solution of tRNA from Escherichia coli (100 ng/μl) [37] to concentration of 0.”
Method
Stable reference genes in granulosa cells of bovine dominant follicles during follicular growth, FSH stimulation and maternal aging
Khan, M. I., Dias, F. C. F., Dufort, I., Misra, V., Sirard, M., & Singh, J. K. (2014) · Reproduction Fertility and Development · doi:10.1071/rd14089 · Cited by 16 · see citing works
Establishment of reference (housekeeping) genes via quantitative real-time PCR for investigation of the genomic basis of abiotic stress resistance in Psammochloa villosa (Poaceae)
Liu, Y. P., Zhang, Y., Liu, F., Liu, T., Chen, J., Fu, G. H., Zheng, C. Y., Su, D. D., Wang, Y. N., Zhou, H. K., et al. (2021) · Journal of Plant Physiology · doi:10.1016/j.jplph.2021.153575 · Cited by 15 · see citing works
Identification of novel genes involved in the biofilm formation process of Avian Pathogenic Escherichia coli (APEC)
Young, M. M., de Oliveira, A. L., Nolan, L. K., Barbieri, N. L., & Logue, C. M. (2022) · PLoS ONE · doi:10.1371/journal.pone.0279206 · Cited by 14 · see citing works
“RT inhibition may also be mediated through specific primer-template interactions [50,51], which would explain why the other primers could bind to the cDNA and exhibit gene expression.”
Background
Standardization of a high-performance RT-qPCR for viral load absolute quantification of influenza A
Pereira, L. A., Lapinscki, B. A., Debur, M. D. C., dos Santos, J. S., Petterle, R. R., Nogueira, M. B., Vidal, L. R., de Almeida, S. M., & Raboni, S. M. (2021) · Journal of Virological Methods · doi:10.1016/j.jviromet.2021.114439 · Cited by 14 · see citing works
Development of one-tube real-time qRT-PCR and evaluation of RNA extraction methods for the detection of Eggplant mottled dwarf virus in different species
Pappi, P. G., Chaintoutis, S. C., Dovas, C. Ι., Efthimiou, K. E., & Katis, N. I. (2014) · Journal of Virological Methods · doi:10.1016/j.jviromet.2014.11.001 · Cited by 12 · see citing works
“his is in accordance with previous investigations which proposed hat the reverse transcriptase can inhibit Taq polymerase, espeially when small quantities of RNA are being tested (Chandler et al., 998; Suslov and Steindler, 2005; Wacker and Godard, 2005).”
Result
DNA-based qualitative and quantitative identification of bovine whey powder in goat dairy products
Zhang, X., Qiao, C., Fu, S., Jiao, Y., & Liu, Y. (2022) · Journal of Dairy Science · doi:10.3168/jds.2021-21618 · Cited by 11 · see citing works
Quantitative analysis of noncoding RNA from paired fresh and formalin-fixed paraffin-embedded brain tissues
Lv, Y., Li, S., Li, Z., Tao, R., Shao, Y., & Chen, Y. (2019) · International Journal of Legal Medicine · doi:10.1007/s00414-019-02210-1 · Cited by 11 · see citing works
Minimization of PCR efficiency differences between standards and samples through dilution of PCR amplicons in reverse transcription buffer
Simard, M., Boucher, É. L., Provost, P. R., & Tremblay, Y. (2006) · Analytical Biochemistry · doi:10.1016/j.ab.2006.12.036 · Cited by 11 · see citing works
“Although reverse transcriptase and dithiothreitol (DTT) were proposed to inhibit PCR reactions [10–13], the observed decrease in PCR efficiency did not arise from these components because both were absent from condition 2.”
Background
Analysis of ADORA2A , MTA1 , PTGDS , PTGS2 , NSF , and HNMT Gene Expression Levels in Peripheral Blood of Patients with Early Stages of Parkinson’s Disease
Семенова, Е. И., Partevian, S. A., Shulskaya, M. V., Rudenok, M. M., Lukashevich, M. V., Baranova, N. M., Doronina, O. B., Doronina, K. S., Rosinskaya, A. V., Fedotova, E. Y., et al. (2023) · BioMed Research International · doi:10.1155/2023/9412776 · Cited by 9 · see citing works
Transcriptome Profiling Reveals Differential Expression of Circadian Behavior Genes in Peripheral Blood of Monozygotic Twins Discordant for Parkinson’s Disease
Семенова, Е. И., Vlasov, I. N., Partevian, S. A., Rosinskaya, A. V., Rybolovlev, I. N., Slominsky, P. A., Shadrina, M. I., & Alieva, A. K. (2022) · Cells · doi:10.3390/cells11162599 · Cited by 9 · see citing works
“After isolation, yeast tRNA was added to the resulting solution of total RNA with a concentration of 1 mg/mL [28].”
Method
Analysis of qPCR Data: From PCR Efficiency to Absolute Target Quantity
Ruijter, J. M., & van den Hoff, M. J. B. (2025) · International Journal of Molecular Sciences · doi:10.3390/ijms262411885 · Cited by 8 · see citing works
“It is also important to note that the presence of a PCR inhibitor or stimulator in the stock solution used to prepare the standard curve will go unnoticed [13], but its dilution will also affect the slope of the regression line and thus the derived PCR efficiency [25,32].”
The impact of analytical quality criteria and data evaluation on the quantification of genetically modified organisms
Meyer, W. A., Caprioara-Buda, M., Jeynov, B., Corbisier, P., Trapmann, S., & Emons, H. (2012) · European Food Research and Technology · doi:10.1007/s00217-012-1787-7 · Cited by 8 · see citing works
“PCR efficiencies above 100 % are an indication for potential inhibition of the polymerase during the amplification process [23].”
BackgroundMethod
Whole-Transcriptome Analysis of Dermal Fibroblasts, Derived from Three Pairs of Monozygotic Twins, Discordant for Parkinson’s Disease
Alieva, A. K., Rudenok, M. M., Novosadova, E. V., Vlasov, I. N., Arsenyeva, E. L., Rosinskaya, A. V., Grivennikov, I. A., Slominsky, P. A., & Shadrina, M. I. (2019) · Journal of Molecular Neuroscience · doi:10.1007/s12031-019-01452-3 · Cited by 8 · see citing works
“Before being added to the reaction mix, cDNA was diluted in an aqueous solution of tRNA from Escherichia coli (100 ng/μl) (Suslov and Steindler 2005) to concentration of 0.02 ng/ μ l .”
MethodInfluential
Reverse transcriptase kinetics for one-step RT-PCR
Rejali, N. A., Zuiter, A. M., Quackenbush, J., & Wittwer, C. T. (2020) · Analytical Biochemistry · doi:10.1016/j.ab.2020.113768 · Cited by 8 · see citing works
Possible Involvement of Genes Related to Lysosomal Storage Disorders in the Pathogenesis of Parkinson’s Disease
Rudenok, M. M., Alieva, A. K., Nikolaev, M. A., Kolacheva, A. A., Ugryumov, M. V., Пчелина, С. Н., Slominsky, P. A., & Shadrina, M. I. (2019) · Molecular Biology · doi:10.1134/s002689331901014x · Cited by 7 · see citing works
Application of an RNA Amplification Method For Reliable Single-Cell Transcriptome Analysis
Suslov, O. N., Silver, D. J., Siebzehnrübl, F. A., Orjalo, A. V., Ptitsyn, A. A., & Steindler, D. A. (2015) · BioTechniques · doi:10.2144/000114331 · Cited by 7 · see citing works
“The phenol/chloroform/isoamyl alcohol (PCI) procedure, followed by ethanol precipitation, removes this negative influence (24).”
Self-citation
Cost-effective optimization of real-time PCR-based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases
Fachmann, M. S. R., Josefsen, M. H., Hoorfar, J., Nielsen, M. T., & Löfström, C. (2015) · Journal of Applied Microbiology · doi:10.1111/jam.12937 · Cited by 7 · see citing works
“However, AEs above 100% was not ranked better than those close to 100% as this also could indicate inhibition leading to an overestimation of the efficiency (Suslov and Steindler 2005).”
Background
Determination of DNA polymerase and nuclease activities of DNA-dependent polymerases using fluorescence detection under real-time conditions
Bragin, A. G., Glushkov, S. A., Ivanov, M., Krasnov, A. A., & Dymshits, G. M. (2008) · Biochemistry (Moscow) · doi:10.1134/s0006297908090083 · Cited by 6 · see citing works
The Application Analysis of Multiplex Real-Time Polymerase Chain Reaction Assays for Detection of Pathogenic Bacterium in Peritoneal Dialysis-Associated Peritonitis
Yang, J., Qi, X., & Wu, Y. (2019) · Blood Purification · doi:10.1159/000495780 · Cited by 6 · see citing works
Novel pan-serotype control RNA for dengue virus typing through real-time reverse transcription-polymerase chain reaction
Álvarez-Díaz, D. A., Quintero, P. A., Peláez‐Carvajal, D., Ajami, N. J., & Usme-Ciro, J. A. (2019) · Journal of Virological Methods · doi:10.1016/j.jviromet.2019.113677 · Cited by 6 · see citing works
Contribution of cell culture, RNA extraction, and reverse transcription to the measurement error in quantitative reverse transcription polymerase chain reaction-based gene expression quantification
Combes, J., Grelier, G., Laversanne, M., Voirin, N., Chabaud, S., Écochard, R., Lasset, C., & Moyret‐Lalle, C. (2009) · Analytical Biochemistry · doi:10.1016/j.ab.2009.06.010 · Cited by 6 · see citing works
Erwinia amylovora bacteriophage resistance
Roach, D. R. (2012) · Brock University Digital Repository (Brock University) · Cited by 5 · see citing works
Manganese-independent Reverse Transcriptase Activity of Tth DNA Polymerase with Two Amino Acid Substitutions
Luo, Z., Xue, Y., Chen, X., Zhang, J., & Lu, C. H. (2023) · Protein and Peptide Letters · doi:10.2174/0929866530666230216113348 · Cited by 4 · see citing works
Differential expression of Dusp1 and immediate early response genes in the hippocampus of rats, subjected to forced swim test
Vlasov, I. N., Filatova, E. V., Slominsky, P. A., & Shadrina, M. I. (2023) · Scientific Reports · doi:10.1038/s41598-023-36611-5 · Cited by 4 · see citing works
Development of RT-qPCR assays for the detection and quantification of three carlaviruses infecting hop
Beaver-Kanuya, E., Wright, A. A., Szostek, S. A., Khuu, N., & Harper, S. J. (2021) · Journal of Virological Methods · doi:10.1016/j.jviromet.2021.114124 · Cited by 4 · see citing works
Development of Low Cost Two-Step Reverse Transcription-Quantitative Polymerase Chain Reaction Assays for Rotavirus Detection in Foul Surface Water Drains
Chung, J. W., Foppen, J. W., & Lens, P. N. L. (2013) · Food and Environmental Virology · doi:10.1007/s12560-013-9111-7 · Cited by 4 · see citing works
“%) is usually seen as an inhibitory effect of the RTase (Suslov and Steindler 2005).”
Background
Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays
Smirnova, E. V., Александрович, Б. К., Barsova, E. V., Varlamov, D. A., Kramarov, V. M., & Ignatov, K. B. (2025) · Methods and Protocols · doi:10.3390/mps8010011 · Cited by 3 · see citing works
Selection and Validation of Stable Reference Genes for RT-qPCR Analyses of Rumex patientia (Polygonaceae) Under Four Abiotic Stresses
Yang, Q., Li, X., Qu, R., Liu, Y., Su, X., Jin, J., Yu, M., Cairang, Z., Zhang, P., Zheng, Y., et al. (2025) · Genes · doi:10.3390/genes16070787 · Cited by 3 · see citing works
“Therefore, we inferred that the presence of inhibitors or pipetting errors can explain the amplification efficiency being greater than 100% [36], but these errors had a limited effect on GAPDH .”
Analysis of LRRN3, MEF2C, SLC22A, and P2RY12 Gene Expression in the Peripheral Blood of Patients in the Early Stages of Parkinson’s Disease
Shulskaya, M. V., Семенова, Е. И., Rudenok, M. M., Partevian, S. A., Lukashevich, M. V., Karabanov, A. V., Fedotova, E. Y., Illarioshkin, S. N., Slominsky, P. A., Shadrina, M. I., et al. (2024) · Biomedicines · doi:10.3390/biomedicines12071391 · Cited by 3 · see citing works
“Then, yeast tRNA was added [29].”
New Developments in Quantitative Real-time Polymerase Chain Reaction Technology
(2014) · Current Issues in Molecular Biology · doi:10.21775/cimb.016.001 · Cited by 3 · see citing works
“Use of polymerases engineered to resist inhibitors As gene expression analysis using RT-qPCR entails the synthesis of cDNA by the reverse transcriptase (RT) enzyme, followed by an exponential amplification of the template by DNA polymerase, any chemical moiety, which is able to alter the fidelity (negatively) of these…”
BackgroundMethod
Single-Cell Gene-Expression Analysis by Quantitative RT-PCR
Warren, L. A. (2008) · Polymer Journal · doi:10.7907/pj42-xb85
Unification of Gene Expression Data for Comparable Analyses Under Stress Conditions
Liu, Z. L. (2011) · Microbiology monographs · doi:10.1007/978-3-642-21467-7_12 · Cited by 3 · see citing works
Specific detection of Malus- and Pyrus-infecting viroids by real-time reverse-transcription quantitative PCR assays
Beaver-Kanuya, E., Szostek, S. A., & Harper, S. J. (2021) · Journal of Virological Methods · doi:10.1016/j.jviromet.2021.114395 · Cited by 3 · see citing works
Quantitative PCR
Overturf, K. (2009) · doi:10.1002/9780813807379.ch3 · Cited by 3 · see citing works
Analysis of Expression of the GRIPAP1, DLG4, KIF1B, NGFRAP1, and NRF1 Genes in Peripheral Blood of the Patients with Parkinson’s Disease in the Early Clinical Stages
Lukashevich, M. V., Rudenok, M. M., Семенова, Е. И., Partevian, S. A., Karabanov, A. V., Fedotova, E. Y., Illarioshkin, S. N., Slominsky, P. A., Shadrina, M. I., & Alieva, A. K. (2024) · Biochemistry (Moscow) · doi:10.1134/s0006297924100092 · Cited by 2 · see citing works
Analysis of the expression of cln3, gabbr1 and wfs1 genes in patients with parkinson’s disease
Starovatykh, Y. S., Rudenok, M. M., Karabanov, A. V., Illarioshkin, S. N., Slominsky, P. A., Shadrina, M. I., & Alieva, A. K. (2020) · Molecular Genetics Microbiology and Virology (Russian) · doi:10.17116/molgen20203802176 · Cited by 2 · see citing works
Development of tools for surveillance of Coxiella burnetii in domestic ruminants and Australian marsupials and their waste
Banazis, M. (2009) · Murdoch University Research Portal · Cited by 2 · see citing works
Screening and stability analysis of housekeeping genes for transcriptomic studies for Danio rerio (Zebrafish) at early developmental stages
Partevian, S. A., Safina, D. R., Rybolovlev, I. N., Rudenok, M. M., Kostrov, S. V., Shadrina, M. I., Slominsky, P. A., & Alieva, A. K. (2024) · Molecular Genetics Microbiology and Virology (Russian) · doi:10.17116/molgen20244202132 · Cited by 1 · see citing works
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“However, one-step RT-PCR protocols are not without inherent limitations and have often been found to be less sensitive than the corresponding two-step protocols [3-5].”

Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR
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Reverse transcriptase inhibits Taq polymerase activity
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Table
Glucose Gene, N., & Cells, M. (2014)

Authors: Suslov, O.
Affiliations: University of Florida (US)
Corresponding author: Suslov, O.
ORCID: Suslov, O.
Venue: Nucleic Acids Research
Publisher: Oxford University Press (OUP)
Published: 2005-11-27
Volume: 33
Issue: 20
Pages: e181-e181
ISSN: 0305-1048 (ISSN-L)
1362-4962
DOI: 10.1093/nar/gni176
PMC: PMC1298932
OpenAlex: W2139173091
Cited by: 121
References: 41
Citations by year: 2026: 5 · 2025: 6 · 2024: 6 · 2023: 5 · 2022: 4 · 2021: 8 · 2020: 8 · 2019: 8 · 2018: 4 · 2017: 3 · 2016: 5 · 2015: 3 · 2014: 8 · 2013: 2 · 2012: 10
FWCI: 1.99
Type: journal article
Language: en
Open access: gold
doi.org
Licence: other-oa
Locations: Nucleic Acids Research (publishedVersion, other-oa)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: National Institutes of Health
National Heart, Lung, and Blood Institute (HL 70143, R01HL070143)
National Institute of Neurological Disorders and Stroke (NS 37556, R01NS037556)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Keywords: reverse transcriptase, PCR inhibition, amplification efficiency, RNA purification, rare transcripts
MeSH terms: Ethanol, Pentanols, Chloroform, DNA, Humans, Chemical Precipitation, Reference Standards, RNA-Directed DNA Polymerase, Reproducibility of Results, DNA, Complementary, Phenol, Reverse Transcriptase Polymerase Chain Reaction, Cell Line, Tumor

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PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency

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