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Archive for the ‘qPCR tutorial’ Category

Real Time Quantitative PCR Software Tutorial

 :: Posted by American Biotechnologist on 01-04-2011

If you are a user or potential users of Bio-Rad’s CFX or MiniOpticon systems be sure to see the message below and the accompanying real time quantitative PCR software tutorial from Bio-Rad’s Sean Taylor, Field Applications Specialist.

I am pleased to provide our recently upgraded version of CFX Manager. Version 2.0 has significant improvements and new tools from the last version 1.6 of the software. I recently did a little test drive and have recorded a 25-minute training video to help you navigate around the new features. Some key highlights include:

1. Scheduling module so that you can schedule your time on the instrument. A great tool for a multi-user instrument.
2. Master Mix Calculator…Just type in your primer concentrations and you get the volumes required for your experiment. No more thinking and more importantly…no more mistakes in volume conversions!
3. Copy and paste wells in the plate editor. I am personally very pleased with this feature.
4. Much easier access to the data you need and very nice and new data view and data export features.

The link to the video is here:
video link

The specifications for the software can be found here:
software specifications

To access the upgrade, you can go to:
upgrade

Click on “Downloads” and you will find:

A. The CFX96 and CFX384 Real-Time PCR Detection Systems Firmware Update.

B. The upgrade patch for CFX Manager 2.0.

C. Precision Melt Analysis™ v1.1 Software Upgrade (This is only required for those who are using Version 1.0 of Precision Melt Analysis Software).

Important: The firmware update comes with detailed instructions that are easy to follow but do not hesitate to call tech support (1-800-4Bio-Rad) if you have any questions about the procedure.

I would like to take this opportunity to wish you and your families health and happiness in 2011!

Sean Taylor
Field Application Specialist
Bio-Rad Laboratories

Click here for other technical resources from Sean including his video entitled “A Practical Approach to MIQE for the Bench Scientist,” A Practical Guide to High Resolution Melt Analysis Genotyping, and the article A Simple Solution to Chromatography for High-Purity Protein Preparations: The Modular Approach

BMC Announces New Topical Series on qPCR

 :: Posted by American Biotechnologist on 09-13-2010

One of our favorite techniques to discuss on the American Biotechnologist is quantitative real-time PCR (qPCR). Previous posts on the topic include:

    That is why we are excited to tell you about Biomed Central’s (BMC) topical series on Quantitative Real Time PCR normalization and optimization, Edited by Joshua S. Yuan. According to BMC, topical series bring together manuscripts published in BMC Research Notes that are associated with individual topics. Through the topical series, BMC Research Notes aims to highlight exciting topical areas of research and provide a home for concise articles that raise awareness and encourage discussion of the subjects covered.

    I’ve scanned through some of the articles posted on BMC’s website and several of them (such as RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts) have piqued my interest. I hope to review some of these articles in this forum over the coming weeks and I welcome your comments and feedback.

    A Practical Approach to MIQE for the Bench Scientist

     :: Posted by American Biotechnologist on 08-11-2010

    In a groundbreaking review published in February 2009, Bustin et al bemoaned the lack of standardization in Quantitative Real-Time PCR (qPCR) experimentation and data analysis. In their critique the authors cite the use of diverse reagents, protocols, analysis methods and reporting formats which has negatively impacted on the acceptance of qPCR as a robust quantitative molecular tool. The most serious technical deficiencies include:

    • sample storage
    • sample preparation
    • sample quality
    • choice of primers and probes
    • inappropriate data and statistical analysis

    In an attempt to correct these problems and instill confidence in the reliability of qPCR, the authors proposed a new set of guidelines which would help standardize the qPCR technique and encourage better experimental practice and interpretation of qPCR results. Since it’s publication, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments or MIQE has become an accepted standard by the scientific community for performing qPCR and the use of the technique is becoming standard practice among molecular biology labs everywhere.

    The MIQE checklist consists of 42 points that cover experimental design, sample quality and preparation, nucleic acid extraction, reverse transcription and qPCR target information. Here’s an illustration which will give you an idea of the comprehensiveness of the list. It’s not meant to be read on the blog. An easy to view checklist can be downloaded from the Real-time PCR Data Markup Language website.

    Sean Taylor et al. subsequently published A Practical Approach to RT-qPCR-Publishing Data That Conforms to the MIQE Guidelines which serves as a great practical guide (as its title implies) for bench scientists. Ironically, the title of the paper does not conform to Bustin’s guidelines, (Bustin suggested replacing the term RT-qPCR with the abbreviation qPCR in order to prevent confusion between the terms “real-time” and “reverse transcription” which both carry the abbreviation “RT”), but nonetheless, the title of the paper should not detract from its valuable content.

    Taylor expands upon Bustin’s organizational approach and provides concrete suggestions for tackling experimental design, RNA extraction, RNA quality control, reverse transcription, primer and amplicon design, qPCR validation, reference gene choice and experimental reproducibility. His suggestions are very brief and to the point and it is definitely worthwhile reading the paper in its original form. Nonetheless, I will try to sum up the salient points below.

    1. Experimental procedures, control groups, type and number of replicates, experimental conditions and sample handling methods should be well defined in advance in order to minimize variability
    2. Handling time should be minimized during the RNA extraction procedure and should include DNase I treatment.
    3. RNA integrity should be assessed for both purity and quality. A pure RNA sample with minimal phonol and protein contamination, will have an OD 260/280 ratio of 1.8 to 2.0 (measured spectrophotometrically). Undegrated/Intact RNA (i.e. RNA quality) will have two sharp bands when run on a formaldehyde agarose gel, with the intensity of the top band being about twoice that of the smaller band. Degraded and impure RNA samples should not be used for qPCR analysis.
    4. Reverse transcription should be performed immediately following the RNA quality control assessment and the same amount of total RNA should be used for each sample. Reverse transcription reaction times should also remain consistent across samples.
    5. Primers should be designed to generate amplicons that are 75-150bp long with no secondary structure and 50-60% GC content. Primer should not have long G or C stretches, should have a G or C cap and should have melting temperatures between 55-65C.
    6. qPCR validation should include: determining the ideal annealing temperature, checking the specificity of the reaction via a melt curve analysis, running a sample for each primer pair on a gel to confirm that the amplicon is the expected size, confirming that the qPCR reaction efficiency is between 90-110% by running a standard curve.
    7. Choosing reference genes (for relative qPCR experiments) that do not exhibit changes in expression between samples from various experimental conditions or time points.
    8. Mitigating biological and technical variability by running at least 3 biological and 2 technical replicates per biological sample for each experiment.

    In addition to being published in the journal Methods, Taylor’s guidelines are also available as a Bio-Rad technical bulletin and can be downloaded here as well.

    Become a Real Time Quantitative PCR Expert

     :: Posted by American Biotechnologist on 07-27-2010

    Real Time Quantitative PCR has become a staple of almost every molecular biology lab in the world. In fact, I doubt that if asked, many scientists would be willing to admit that they don’t do real time quantitative PCR. The cost of attaining a Real Time qPCR instrument and the per assay costs have come down exponentially putting real time quantitative PCR within the reach of most molecular biology labs.

    In this video tutorial by Bio-Rad Laboratories (which is applicable to ALL real time quantitative PCR platforms), you will learn the basics of Real Time qPCR and the science behind absolute and relative quantification methods. Absolute quantification is used when looking to answer the question of “how many” or “how much.” Examples include how many copies of gene x are in my sample or how much viral load is there in a given biological sample. Relative quantification, (the more popular technique for data analysis), is used when looking to measure changes or differences in gene expression between various samples. Examples include looking for upregulation or downregulation of a gene in a normal versus disease state or changes in gene expression following an siRNA gene silencing experiment.

    Several methods have been published for conducting real time qPCR data analysis using relative quantification methods including Livak, delta CT and the Pfaffl methodology. The video will also explain the differences between each of these techniques and a review of their uses and limitations.

    If you are very ambitious, visit the original Bio-Rad video link for a clearer picture that combines both of these youtube videos into one (youtube doesn’t allow for videos that are over 10 minutes long and tends to make text a bit blurry).

    Bio-Rad Tutorial: Validating a SYBR Green I Assay

     :: Posted by American Biotechnologist on 05-26-2010

    In this latest tutorial by the Bio-Rad Technical Support Team you will learn how to validate a SYBR Green I assay for real-time quantitative PCR (qPCR). It is important to validate all qPCR assays prior to commencing your experiment in order to confirm that your reactions will be efficient, specific, sensitive and reproducible. SYBR Green I assay validation should also be done when using a new set of primers or a new DNA template/primer combination regardless of the chemistry that you intend to use in your actual experiment.

    Hats off to Linda Lingelbach and Kathy Silvey of Bio-Rad Technical Support who worked hard at putting this enlightening video together.

    Prior to watching this tutorial, it is recommended that you watch the qPCR-Hallmarks of an optimized SYBR Green I assay first.