Dataset Information

Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons.

ABSTRACT: Analyzing rare DNA and RNA molecules in limited sample sizes, such as liquid biopsies and single cells, often requires preamplification, which makes downstream analyses particularly sensitive to polymerase chain reaction (PCR) generated contamination. Herein, we assessed the feasibility of performing Cod uracil-DNA N-glycosylase (Cod UNG) treatment in combination with targeted preamplification, using deoxyuridine triphosphate (dUTP) to eliminate carry-over DNA. Cod UNG can be completely and irreversibly heat inactivated, a prerequisite in preamplification methods, where any loss of amplicons is detrimental to subsequent quantification. Using 96 target assays and quantitative real-time PCR, we show that replacement of deoxythymidine triphosphate (dTTP) with dUTP in the preamplification reaction mix results in comparable dynamic range, reproducibility, and sensitivity. Moreover, Cod UNG essentially removes all uracil-containing template of most assays, regardless of initial concentration, without affecting downstream analyses. Finally, we demonstrate that the use of Cod UNG and dUTP in targeted preamplification can easily be included in the workflow for single-cell gene expression profiling. In summary, Cod UNG treatment in combination with targeted preamplification using dUTP provides a simple and efficient solution to eliminate carry-over contamination and the generation of false positives and inaccurate quantification.

SUBMITTER: Andersson D

PROVIDER: S-EPMC6214100 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons.

Andersson Daniel D Svec David D Pedersen Cathrine C Henriksen Jørn Remi JR Ståhlberg Anders A

International journal of molecular sciences 20181016 10

Analyzing rare DNA and RNA molecules in limited sample sizes, such as liquid biopsies and single cells, often requires preamplification, which makes downstream analyses particularly sensitive to polymerase chain reaction (PCR) generated contamination. Herein, we assessed the feasibility of performing Cod uracil-DNA <i>N</i>-glycosylase (Cod UNG) treatment in combination with targeted preamplification, using deoxyuridine triphosphate (dUTP) to eliminate carry-over DNA. Cod UNG can be completely a ...[more]

PMID: 30332749

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Project description:BackgroundDue to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan(R) PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp).ResultsT-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 +/- 0.33 Ct values. The coefficients for inter- (1.89 +/- 0.48%) and intra-assay variation (0.85 +/- 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 +/- 0.33, without significant differences between self-designed and ABI inventoried Taqman(R) gene assays. Only two of the tested Taqman(R) ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 +/- 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 +/- 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 +/- 0.74), albeit higher inter-assay CVs (5.38 +/- 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.ConclusionIn comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

Dataset Information

Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons.

Publications

Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons.

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