ArticleBMC molecular biology2015
Pre-amplification in the context of high-throughput qPCR gene expression experiment.
Article in BMC molecular biology, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 24 papers.
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Who cites it
24 citing papers in PubMed, 48 citations in OpenAlex.
- MicroRNA profiling in plasma samples using qPCR arrays: Recommendations for correct analysis and interpretation.PloS one · 2018Trial
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- High-Throughput Microfluidic Quantitative PCR Platform for the Simultaneous Quantification of Pathogens, Fecal Indicator Bacteria, and Microbial Source Tracking Markers.ACS ES&T water · 2023Article
- Transcriptional Biomarkers and Immunohistochemistry for Detection of Illicit Dexamethasone Administration in Veal Calves.Foods (Basel, Switzerland) · 2022Article
- Eight weeks of combined exercise training do not alter circulating microRNAs-29a, -133a, -133b, and -155 in young, healthy men.European journal of applied physiology · 2022Article
- Gene Expression Studies in Formalin-Fixed Paraffin-Embedded Samples of Cutaneous Cancer: The Need for Reference Genes.Current issues in molecular biology · 2021Article
- Gene Expression Signature Associated with Clinical Outcome in ALK-Positive Anaplastic Large Cell Lymphoma.Cancers · 2021Article
- Tutorial: Guidelines for Single-Cell RT-qPCR.Cells · 2021Review
- An improved method for specific-target preamplification PCR analysis of single blastocysts useful for embryo sexing and high-throughput gene expression analysis.Journal of dairy science · 2021Article
- Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs.Biochemia medica · 2021Article
- Design of a High-Throughput Real-Time PCR System for Detection of Bovine Respiratory and Enteric Pathogens.Frontiers in veterinary science · 2021Article
- Estimating Clinically Relevant Cut-Off Values for a High-Throughput Quantitative Real-Time PCR Detecting Bacterial Respiratory Pathogens in Cattle.Frontiers in veterinary science · 2021Article
- Development of a high-throughput real-time PCR system for detection of enzootic pathogens in pigs.Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc · 2020Article
- Validation of molecular biomarkers for preoperative diagnostics of human papillary thyroid carcinoma in fine needle aspirates.Gland surgery · 2019Article
- Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples.BMC molecular biology · 2018Article
- Article
- Triage of high-risk HPV-positive women in population-based screening by miRNA expression analysis in cervical scrapes; a feasibility study.Clinical epigenetics · 2018Article
- Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform.Frontiers in cellular and infection microbiology · 2018Article
- Resolving Species Level Changes in a Representative Soil Bacterial Community Using Microfluidic Quantitative PCR.Frontiers in microbiology · 2017Article
Corrections and comments
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Authors and funding
8 authors at 4 institutions in 4 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundWith the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm).
resultsFor evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted.
conclusionsWe set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.