Posts tagged ‘Real-Time PCR’
Bio-Rad Laboratories, Inc. today announced the launch of its SsoAdvanced™ PreAmp Supermix and PrimePCR™ PreAmp Assays (PrimePCR PreAmp Assays are available in the U.S., Canada, Australia, New Zealand, Singapore, and E.U. only). The supermix is formulated to permit unbiased, target-specific preamplification of cDNA or genomic DNA (gDNA), which allows researchers to stretch their limited nucleic acid samples. PrimePCR PreAmp assays are wet-lab validated preamplification primers that work seamlessly with the supermix to ensure optimal preamplification for real-time PCR.
The SsoAdvanced PreAmp Supermix is especially useful for researchers who are working with limited samples or are interrogating multiple targets simultaneously. It enables simultaneous 1,000-fold target-specific preamplification of as little as 100 pg of cDNA (or gDNA) for up to 100 genes while delivering the industry’s lowest bias. It is also more cost-effective than any other preamplification solution available.
The SsoAdvanced PreAmp Supermix can be paired with Bio-Rad’s iScript™ Advanced or iScript Supermix reverse transcription reagents. Using the PreAmp Supermix preamplification protocol (see illustration), researchers first isolate RNA and synthesize cDNA using either of the iScript reagents. After mixing the cDNA (or gDNA) with SsoAdvanced PreAmp Supermix and pooled primers (or with PrimePCR PreAmp assays), PCR is performed to preamplify targets of interest. The reaction is then ready for real-time PCR amplification and detection.
For the most accurate gene expression analysis, researchers should pair the SsoAdvanced PreAmp Supermix with PrimePCR PreAmp assays, which are available for every PrimePCR gene expression assay. PrimePCR assays are the only wet-lab validated and specificity-verified primers. The SsoAdvanced PreAmp Supermix is validated for SYBR® Green, probe-based (e.g. TaqMan®), and custom-designed real-time PCR assays.
Real time PCR for quantitative analysis of gene expression has become an integral part of many (if not most) research labs around the world. When preparing for qPCR analysis, it is important to choose reference genes than do not vary under experimental conditions and that can be used to normalize your data to accurately reflect changes due to the given treatment. (See: Are you using the right reference genes?)
Choosing the right reference genes can be a very difficult task. Most experts agree that when performing qPCR for gene expression, the more reference genes used, the merrier. Unfortunately, “the more the merrier” is also associated with “the more, the more complicated.”
Fortunately, Dr. Sean Talyor, Field Application Specialist at Bio-Rad Canada has found a great program that makes use of many online databases to help you find the ideal reference genes. In the following tutorial, Sean will show you how to use the Refgenes program for choosing your ideal reference genes. The program utilizes a massive microarray database to help determine gene expression under a variety of experimental conditions.
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 as 18S? Check out this article on suitable reference gene selection 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.
Fu, L., Jia, H., Dong, Q., Wu, J., Zhao, Y., Zhou, H., Ren, N., Ye, Q., & Qin, L. (2009). Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses BMC Cancer, 9 (1) DOI: 10.1186/1471-2407-9-49
Classifying and understanding genetic variation between populations and individuals is an important aim in the field of genomics. Many common diseases (diabetes, cancer, osteoporosis, etc.) and clinically relevant phenotypic traits are elicited from the complex interaction between a subset of multiple gene products and environmental factors. High resolution melt (HRM) analysis is the quantitative analysis of the melt curve of a DNA fragment following amplification by PCR and can be considered the next-generation application of amplicon melting analysis. It is a low-cost, readily accessible technique that merely requires a real-time PCR detection system with excellent thermal stability and sensitivity and HRM-dedicated software. However, careful sample preparation and planning of experimental and assay design are crucial for robust and reproducible results. The following guidelines assist in the development of such assays.
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Bio-Rad Laboratories’ recently launched its newest 2-colour real time PCR machine. The CFX Connect real-time PCR detection system offers two-target analysis, honey comb block uniformity, thermal gradient optimization and solid state optical technology.
For more information on the system check out the CFX Connect brochure .
Click here for step-by-step instructions on how to setup data using CFX manager software.
In this slideshare presentation, you will be introduced to the basics of High Resolution Melt Analysis including its applications, important considerations, assay design and optimization and a tutorial on Precision Melt Analysis Software. The presentation is completely self-navigable and can be reviewed at your own pace. If you would prefer a video presentation of this material please see the HRM video tutorial. Also be sure to read High Resolution Melt Parameter Conditions for Optimal Data Resolution and A Practical Guide to High Resolution Melt Analysis Genotyping.
We have written several posts about the utility of the iPhone in medical research (see: Science with the iPhone, NCBI Blast Using an iPhone and RFID Technology, and The Bio-Rad Real-Time PCR iPhone Application). Now there is a MIQE qPCR app for all ipod/iphone users to add to their arsenal.
The MIQE app helps scientitst review scientific work and check their own project’s MIQE compliance. Plus, the app includes a list of the most current qPCR news and events and “emergency” contact numbers that you can call/email should you have any questions about your qPCR experiments.