mybiogazelle

1. Can I analyze data from my qPCR instrument in qbase^PLUS?

In principle, qbase^PLUS can read data export files from any real-time PCR instrument software, as long as the user organizes the data into a format currently accepted by qbase^PLUS. To this purpose, 3 general and instrument independent formats are available (simple, qBase, RDML). In addition, qbase^PLUS directly reads export files (containing Cq values, not the raw (fluorescent) data or binary files) from the following instruments:

Guidelines and example files can be found in the data import section.

2. What are the hard and software requirements for running qbase^PLUS?

qbasePLUS 2.0 makes use of a completely rewritten calculation engine, making it ideal for high-throughput real-time PCR data-analysis on standard computers. We recommend at least 1 Gb RAM memory and a Pentium IV, Athlon or equivalent processor.

qbasePLUS is compatible with Microsoft Windows XP and above with a recent version of JAVA (at least 1.5 for qbasePLUS 2.0 and 1.6 for novel releases). qbasePLUS is also compatible with Linux and MAC OS X. For the latter at least 10.4 (Tiger) is required (10.5 - Snow Leopard for qbasePLUS releases after 2.0).

3. How do I install qbase^PLUS?

In Windows, you need administrator installation rights and the most recent version of Sun's Java. qbase^PLUS installation starts automatically after double cliking on the downloaded installer file.

For Mac, Java comes preinstalled. Just install qbase^PLUS using administration rights.

For Linux, there is no installer. Download and extract qbase^PLUS to an appropriate folder (e.g. /usr/local/qbaseplus). Set the execution bit for qbase^PLUS ('chmod 001 qbaseplus' on the command line, or file properties in the graphical user environment).  qbase^PLUS requires Sun Java to operate; you can get it using your package manager, or from http://www.java.com/en/download/linux_manual.jsp.

More information about the installation and upgrade procedure can be found here.

4. Where can I find my install ID?

In qbase^PLUS, go to Help > Licensing > Request demo license. Please note that the install ID is computer specific; a license key generated using this install ID will only work on the computer that generated the install ID.

More information about the installation and upgrade procedure can be found here.

5. Where is the user manual?

The software comes with dedicated chapters explaining the most important features: data import, run annotation, user interface, efficiency correction, genormPLUS, quality control, statistics, copy number analysis, and global mean normalization. There is also a quick guide that describes how quantitative PCR data can be imported and analyzed in qbase^PLUS. All documents are available in the support section of the website.

Biogazelle also organizes webinars on a regular basis, civering different topics.

Do not hesitate to contact us if you need personal support (only for users with a basic and premium license).

6.How to activate and install a purchased license key?

You need to log in to your private mybiogazelle space, and navigate using the left menu to 'my licenses' and 'license overview'. Activate your license key by clicking on it and providing your install ID (see FAQ 'Where can I find my install ID'). After activation, you can download your license key (*.lic file) and unlock qbase^PLUS by going to Help > Licensing > Install license, and browsing to the location on your hard disk or other medium where you saved the license key.

More information about the installation and upgrade procedure can be found here.

7. Why do I have to activate my license key?

All qbase^PLUS license keys are specific for a given computer. During activation, computer specific information is embedded in the encrypted license key. This is done by integrating your install ID (see FAQ 'Where can I find my install ID') that is generated by qbase^PLUS after installation on your computer. The license key can therefore only be installed on the computer used for generation of the install ID.

More information about the installation and upgrade procedure can be found here.

8. How do I migrate from qBase to qbase^PLUS?

Using the qBase Run Extraction tool, you can export all your former Microsoft Excel qBase experiments into individual annotated qBase run files. All files belonging to the same experiment can be simultaneously imported in qbase^PLUS using the batch import mode (just select multiple files for import) and selecting 'qBase' as import file format.

9. What is inter-run calibration and how does it work?

Inter-run calibration is a calculation procedure to detect and remove (often underestimated) inter-run variation. Detailed information is available in Hellemans et al., Genome Biology, 2007. The basic principle is that the experimenter measures one or (preferentially) more identical samples in different (to be calibrated) runs (in conjunction of course with many other samples that actually are different across the runs). These identical samples (so-called inter-run calibrators) can then be used to detect and correct inter-run variation.

qbase^PLUSis the only software that (1) allows inter-run calibration using more than one inter-run calibrator (making it more accurate), (2) that performs inter-run calibration after normalization (allowing the experimenter to re-synthesize cDNA from the inter-run calibrator RNA samples), and (3) that propagates the error introduced during the inter-run calibration procedure. A dedicated manual chapter is available that explains how to calibrate runs in qbase^PLUS.

10. How does qbase^PLUS deal with replicate measurements?

qbase^PLUS automatically deals with technical replicates or repeated measurements. These are recognized as different PCR wells with an identical sample and target name. The Cq values of all (non-empty) wells of a replicate group are averaged at the very beginning of the calculation workflow. Of course, outliers can be removed before calculations.

As of version 2.0, it is also possible to define biological replicates. A dedicated manual chapter (sample grouping) is available that explains how to to deal with biological replicates in qbase^PLUS.

A third type of replicate measurements are the so called inter-run calibrators. These are identical samples that are measured for the same target in different runs (in order to detect and correct inter-run variation, see also Frequently Asked Question "What is inter-run calibration and how does it work?"). To avoid interpretation of inter-run calibrators as technical replicates, they should have a different sample name in the different runs (e.g. IRC1_a, IRC1_b, ...).  Users should then indicate that a number of sample names actually refer to the same biological sample that is used as an inter-run calibrator (e.g. both IRC2_a and IRC2_b refer to the second inter-run calibrator, sample IRC2). This procedure is known as setting the inter-run calibrators (more info in the inter-run calibration manual).

11. How to flag bad technical replicates based on a standard deviation threshold?

qbase^PLUS flags bad replicates based on a user defined maximum allowed difference in Cq values (defined in the Experiment quality control settings). Of note, there is a clear relation between the standard deviation and difference in Cq (independent of the actual Cq values). In fact, the standard deviation increases 0.1 units per 0.14142 cycle difference between duplicated reactions; similarly, the standard deviation increases 0.07071 units per 0.1 cycle difference between duplicated reactions. Hence, if you want to flag bad duplicates that differ by more than 0.2 standard deviations, you need to use a 0.28284 cycle difference. For triplicates, a standard deviation threshold of 0.1 means that the highest and lowest Cq value can only differ by maximum 0.1 Cq values from the middle point.

More information in "The Importance of Quality Control During qPCR Data Analysis" published in International Drug Discovery August/September 2010.

14. How is PCR efficiency calculated and used for relative quantification?

The PCR efficiency E can be calculated from the slope of a serial dilution as follows: E = 10^(-1/slope) - 1 (with an E of 1 being perfect, indicating 100% efficiency). The formula to go from Cq values to relative quantities is (E+1)^delta-Cq (hence 2^delta-Cq for an assay with 100% PCR efficiency).

The gold standard method for PCR efficiency estimation is a serial dilution of representative template (e.g. a mixture of RNA or cDNA from your samples). Nevertheless, there are a few algorithms (from the large number out there) that seem to provide a reliable estimate of the PCR efficiency based on a single amplification curve. Importantly, the caclulated results should be precise and accurate (and many algorithms fail in this respect). Hence, various papers (see references below) point at the danger of using sample specific PCR efficiencies based on a single amplification curve (or even replicate measurements). The authors rather propose to average the sample specific efficiencies to obtain a target (gene) specific efficiency. This is also what Biogazelle recommends to its qbase^PLUS users.

In qbase^PLUS, users should provide the base of the exponential function as amplification efficiency value for relative quantification if they want to correct for target specific efficiency. The base number is the E value + 1, e.g. 1.95 for 95% efficiency (E value of 0.95).

References: Nordgård et al., Anal Biochem, 2006; Goll et al., BMC Bioinformatics, 2006; Karlen et al., BMC Bioinformatics, 2007.

15. What signifies a CNRQ value in the result table?

CNRQ means Calibrated Normalized Relative Quantity (see Hellemans et al., Genome Biology, 2007). If one does not perform inter-run calibration, then CNRQ equals NRQ and is the PCR efficiency corrected target of interest relative quantity (RQ) divided by the geometric mean of the corrected reference target RQs. If one uses only one reference target (not recommended), then the target of interest RQ is divided by the reference target RQ.

16. What to do if you want to transfer qbase^PLUS to a new computer?

qbase^PLUS license keys are linked to a specific computer. You need to contact Biogazelle if you want to transfer your license from one computer to another. Send the installation-ID of both your old and your new computer to info@biogazelle.com and Biogazelle arranges for a new license key.

17. How to get further support?

If you need specific information, or run into a problem, first check the manual chapters that illustrate most functionalities in qbase^PLUS. If this does not help, please go over the Frequently Asked Support Questions (see higher). If all fails, please use the dedicated Support form that is available from your personal 'my support' page after logging in to the Biogazelle website (mybiogazelle section top right). Provide as much information as possible to facilitate trouble shooting. This includes a description of what you were trying to achieve and what you were doing before the problem occurred.  If an error occurred, qbase^PLUS will store relevant information in a log file. The content of this log file is available in the Error Log view (Window > Show View > Error Log) by clicking the Export Log icon. When reporting an error, please upload this file.