AccuPower® qPCR Array Service

qPCR Array Service is qPCR analysis service that follow the guideline of MIQE† (Minimum Information for Publication of Quantitative Real-Time PCR Experiments), which are guidelines for qPCR experiments for publishing data.

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Cat. No.
S-6001

Overview

  • Bioneer's qPCR Array Service provides qPCR analysis services that comply with the *MIQE guidelines, which are guidelines for Real-Time PCR experts and qPCR experiment guidelines for paper publication.

    *The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) “qPCR gene expression analysis” Agreed for quality control of papers: Bustin, S.A., et al. 2009. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry 55:4, 611-622.

Features and Benefits

  • All-in-one service providing from nucleic acid extraction to qPCR analysis
  • Accurate and rapid high-quality qPCR analysis service
  • Customized-qPCR analysis service
  • Save time and cost for Primer set up
  • NGS result verification – qPCR validation

Service list

According to the type of requested samples, we first choose the suitable high quality, high purity RNA extraction kit to undergo total RNA extraction. Moreover, to eliminate gDNAs that affects mRNA quantification, we treat the sample with RNase free DNase I.

All the impurities in the extracted total RNA may affect the amplification efficiency by inhibiting the PCR polymerase reaction. Therefore, spectrophotometers are used for measuring both the quantity and purity of the nucleic acids. Since it is difficult to accurately quantify the amount of degraded RNA, capillary gel electrophoresis is also used to measure the RNA quality score (RQS). All those measurements will be recorded in the report and sent to the customers. We will proceed to the next step only if the purity and the degree of degradation of the RNA satisfy the recommended value (RQS > 8.0).

*If the customer wants, the qPCR service will be still available for samples even for those that do not satisfy the RQS standards. However, Bioneer will not guarantee the reliability of the derived data.


Figure 1. Total RNA quality check example. Agilent 5200 Fragment Analyzer Results.

Target-specific primers are designed using primer blast and bioinformatics tool. They are designed to amplify all transcript variants of the target gene, and self primer-dimer forming sequences are excluded. After Primers that are designed and synthesized validate target gene specific amplification using standard template such as Stratagene reference total RNA (Human, Mouse, Rat), use in qPCR.


Figure 2.  (A, B) Identification of target gene amplification and single melting peak using Exicycler™ 96 Real-Time Quantitative Thermal Block. (C) Identification of single band in Gel electrophoresis.

cDNA synthesis is an important step along with RNA quality check for the gene expression analysis as its efficiency is greatly influenced by the mRNA. By using Bioneer’s patent technologies of Thermostable RTase (RocketScript™) and Cyclic Temperature Reverse Transcription (CTRT), full-length cDNA can be synthesized, even with RNAs having complex secondary structures, for accurate expression analysis. The RT primers used may vary from Oligo dT and Random primer(dN6, dN12) depending on the purpose of the customer’s research purposes.


Figure 3. Comparison of cDNA synthesis efficiency between Bioneer's and other companies’.

Bioneer's qPCR Array Service is performed by experienced experts in qPCR experiments. It is a high-sensitivity detection optical system and has been patented Exicycler™ 96 Real-Time Quantitative Thermal Cycler is used to produce highly reliable qPCR data. All equipment is regularly inspected and managed by ISO regulations.


Figure 4.  Exicycler™ 96 Real-Time Quantitative Thermal Block, Exicycler™ 384 Real-Time Quantitative Thermal Block, Amplification curve.

Analyzing and providing qPCR results

2-ΔΔCt method is used for carrying out relative quantitative analysis using qPCR. Excel file will be sent to the customers with the following data: raw Ct values, melting curve analysis, p-value, and fold change. (additional providable data: scatter plots, volcano plots, and heat map image.) qPCR raw data can be provided if Exicycler™ analysis software is installed. Information on primers such as genetic information, amplicon size, and PCR conditions following the MIQE can be also provided upon the customer’s request.


Figure 5.  qPCR analysis results. (A) Fold change. (B) Heat map. (C) Scatter plot. (D) Volcano plot.

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