Posts Tagged ‘Real-Time PCR’

Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay

 :: Posted by American Biotechnologist on 02-16-2010

If you are currently doing quantitative PCR (qPCR) or plan on beginning qPCR experiments you MUST check out this latest video tutorial from Bio-Rad’s tech support group.

This tutorial describes the characteristics of an optimized SYBR green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks of an optimized qPCR assay.

qpcr syber tile

The Versatility of High-Resolution Melt Analysis

 :: Posted by American Biotechnologist on 01-19-2010

High-Resolution Melting Analysis (HRMA) is rapidly becoming a very attractive tool to not only to screen patients for pathogenic variants but also for presequence screening, single nucleotide polymorphism (SNP) typing, methylation analysis, quantification (copy number variants), an alternative to gel-electrophoresis and clone characterization. The ease of use, simplicity, flexibility, low cost, nondestructive nature, superb sensitivity, and specificity of HRMA has brought the technique to the forefront of life science research.

I did some literature searching and have attached a recent review that covers the topic very well. The article was written prior to Bio-Rad’s launch of our own HRMA software module integrated into the CFX96 and CFX384 instruments so I have also attached a flyer on the topic which will show you the accessibility to HRMA through these instruments, associated software and SsoFast EvaGreen Supermix.

High Resolution Melt Analysis – Review Article 2009

Precision Melt Analysis Software Bulletin_5798A

Sean Taylor, pHD
Field Applications Specialist
Bio-Rad Laboratories

Lab Automation with Style

 :: Posted by American Biotechnologist on 01-07-2010

As seen in Genomeweb this week, Bio-Rad Laboratories launched an automated front end for its CFX96 and CFX384 real-time PCR detection systems, marking the company’s first automated real-time PCR system. The new technology, called the CFX automation system, includes a bench-top plate handler that can load up to 20 384-well plates, or 7,680 samples, at one time on the CFX384. It also includes control software that manages the configuration and operation of the CFX automation system so that researchers can assign either the same or manifold PCR protocols across each plate. The automated system is targeted at anyone who wants to increase their throughput and run their system in an automated environment.

Essentially, we are talking about having a set of machines that will pipette samples and mastermix into the PCR plate, load the plate into the PCR machine, run the protocol and EMAIL THE DATA to you once the run has completed.

Remember the days of pipetting into centrifuge tubes, setting up 3 waterbaths (one for denaturing, another for annealing and another for extending) and then manually incubating the tubes in each bath for 1 minute and cycling that 30-40 times? (OK…so I’m not that old to remember that either but my master’s degree supervisor kept talking about how he did that when he was in the lab). In any event, doing research used to mean that we’d have to be in the lab setting up our experiments and waiting for our results. How cool is it that I can now set up my reagents, primers probes and samples, walk away, go home and do my analysis from home once the data file is sent to me? This is definitely the wave of the future. Couple this with a laboratory management program such as bioKM from Biodata that could organize your data within the context of your experiment and all you’d be missing is the automatic “peer-reviewed” paper writing machine.

Very cool. What will they think of next!

High Resolution Melt Analysis Applicatons

 :: Posted by American Biotechnologist on 12-29-2009

Genomics is rapidly expanding beyond merely identifying the genetic makeup of organisms to encompass studies of how variations in genotype impact physiological functions. See the article below which was published in the October 15th edition of Genetic Engineering and Biotechnology news to learn more about high-res met analysis as an alternative to probe-based genotyping.
High-Resolution Melt Analysis Applications

Are you using the right reference genes?

 :: Posted by American Biotechnologist on 12-10-2009

Considering Real-Time PCR for gene expression analysis. Have you tested multiple reference genes or are you just going to run with your favorite such ast 18S? Check out this important article before you move forward. It could save you lots of grief in the long run.

Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses
Li-Yun Fu et al. BMC Cancer 2009, 9:49

Housekeeping genes are routinely used as endogenous references to account for experimental differences in gene expression assays. However, recent reports show that they could be de-regulated in different diseases, model animals, or even under varied experimental conditions, which may lead to unreliable results and consequently misinterpretations. This study focused on the selection of suitable reference genes for quantitative PCR in human hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) with different clinical outcomes.

Suitable Reference Gene Selection 2009