Page 2 - Gene Expression Profiling

Hepatic stellate cells have recently gained a great deal of attention regarding their contribution to the progression of diseases such as liver fibrosis, non-alcoholic steatohepatitis (NASH), and hepatocellular carcinoma. They are mesenchymal cells that are located between sinusoidal endothelial cells and hepatocytes in the space of Disse or perisinusoidal space and represent about 5-10% of cells in the liver. Hepatic stellate cells play a critical role in liver homeostasis and perform a diverse set of functions, some of which are poorly understood. In a normal healthy liver, stellate cells are quiescent and store vitamin A droplets.  Additionally, stellate cells are involved in vasoregulation, monitoring extracellular matrix deposition, and the production of factors that stimulate hepatocyte regeneration.

In response to liver damage, stellate cells receive signals from hepatocytes, hepatic sinusoidal endothelial cells, and immune cells to activate.  Once given the signal to activate,

qPCR is a powerful tool for quantification of gene expression levels and copy number variation. Despite the advances in next-generation sequencing (NGS), qPCR still serves as the "gold standard" for gene expression analysis. Due to poor reproducibility and vast lab-to-lab variation, all NGS data requires qPCR validation. However, as essential as qPCR is, qPCR loading can be challenging.

Aging and Telomere Length Quantification by qPCR

Aging is a time-dependent decline of the body’s functional capabilities and is an inevitable course of life (as shown in the image below, extracted from the Wall Street Journal). The rate of aging though is highly variable among individuals. When evaluating health status, the virtual (biological) age and actual (chronological) age are both important. For this reason, scientists have long searched for reliable biomarkers of aging to determine biological age.  Currently, there is no well-accepted aging biomarker that has been identified.

The 2017 Nobel Prize in Physiology or Medicine was awarded to Jeffrey C Hall, Michael Rosbash, and Michael W Young for research that established key mechanistic principles on how circadian rhythms are regulated. Circadian rhythms are endogenous oscillations adjusted to changing external cues and driven by circadian clocks.

All multicellular organisms and almost all tissues in the body regulate circadian rhythms using a similar mechanism as the one elucidated by the 3 Nobel Laureates. Indeed, a majority of the genes expressed in our bodies are regulated by our circadian clock, consequently requiring careful calibration of our physiology with our environment. For example, the cell cycle is regulated by the circadian clock, and disruptions in circadian rhythms can be associated with the sort of aberrant cell cycling associated with tumorigenesis such as breast cancer.  Circadian biology as it pertains to overall health, disease, and disease susceptibility has grown into a vast research

Next generation sequencing (NGS), such as Whole Exome Sequencing (WES) and RNA-Sequencing (RNA-Seq), provides a high throughput approach for DNA sequencing and gene expression analysis. It is rapidly advancing our knowledge on almost all aspects of genetic research and shedding light on how individual or groups of genes may regulate biological processes. Powerful as NGS is, however, many high impact journals require that NGS data include qPCR validation.

So why is qPCR validation still such an important step? There are 3 main reasons.

First, compared to qPCR, NGS is considerably more complicated and consequently the potential for errors is increased. Reproducibility can also be a problem due to the complexity of NGS experiments.

Second, there can be intrinsic biases in NGS. For one, due to the random sampling nature of NGS, the sensitivity is largely based on the "sequencing depth", or how many times the template gets read. If the transcripts are expressed at low levels, it may not reach

3 Reasons Why You Should Take "Pseudogenes" Seriously in Your Research

Protein-coding DNA, or “functional” genes, only accounts for up to 2% in the human genome. Until recently, the remaining 98% have been referred to as “junk DNA” for their putative noncoding feature. The name itself implies that junk DNA is useless and can be ignored. Further research, however, has shown that junk DNA may actually be crucial in the functionality of our genome. For instance, have you ever wondered why human astrocytes and hepatocytes originated from the same fertilized egg, and carry exactly the same genetic information, yet they differ greatly in their development, morphology and functions? While part of the reason lies in epigenetics, where gene expression is regulated by epigenetic modification such as DNA methylation, some researchers have also been searching for clues in the so-called junk DNA. As a result, "pseudogenes" were identified, and their diverse roles in maintaining normal cell biology

Cancer is a collection of over 200 diseases where the only common denominator is rogue cells^1,2.  The ways in which a cell can go rogue is so varied that cancer has its own separate biology where order and normalcy are not readily apparent.  Cancer does not even have to be solid.  Indeed, blood cancers like leukemia and lymphomas account for about 10% of new cancer diagnoses in the US³.  Our understanding of cancer is continually being refined, and in preparation for our visit to the 2017 American Association for Cancer Research (AACR) conference, this blog post will give a brief overview of human history with cancer, highlight some accomplishments in cancer research, and discuss two future directions for cancer therapy research.

Fossilized bones and mummies of ancient Egypt provide some of the earliest evidence of cancer, and the first recorded description of cancer dates back to circa 3000 BC characterizing breast tumors as a disease for which “there is no treatment⁵.”

Online webinar hosted by sciencell to learn tips for successful qPCR, a technique for gene expression profiling

CARLSBAD, CALIFORNIA, USA, -- Sciencell Research Laboratories, Inc. , a global provider of primary cells, cell culture media and reagents for life science industry, announced today that it will host a live, complimentary webinar titled, “Technical Tips for Successful qPCR : WEBINAR SERIES”, on Tuesday, January 24, from 1:00 PM to 2:00 PM Pacific Time. The webinar will offer expert overview on successful qPCR from Dr. Yongjiang Daniel Li, Research Scientist, Research and Development, Sciencell Research Laboratories, Inc., with assistance from Dr. Jennifer Welser, Scientific Affairs, Sciencell Research Laboratories, Inc.
“qPCR array provides a quick, accurate and sensitive approach for gene expression profiling. Though powerful and widely-used, attaining reliable results from qPCR can be difficult.” explains Daniel. “In this webinar we will discuss unique tips for primer design,

SAN DIEGO, Calif. -- ScienCell Research Laboratories, Inc. recently announced it would be expanding its cell culture products to the gene expression profiling market. ScienCell, who prides itself on supplying unique primary cell types for nearly 20 years, will now supply qPCR array kits and individual primers to study gene expression profiles. "We are glad to offer validated qPCR gene expression analysis products so that researchers can spend their time on more important aspects of their studies," said mastermind behind GeneQuery™ (https://sciencellonline.com/en/products-services/genetics-genomics/), Yongjiang Daniel Li, Ph.D. "Our team is dedicated to designing unique and quality qPCR array kits that are focused on primary cell development and functions, cancer research, hereditary diseases, and biomarker discovery."

Primary cells are increasingly used by researchers as an in vitro model of in vivo biological processes. As the leading primary cell provider, ScienCell now enables researchers

Cell-based assays are widely used in basic and translational research as cost-effective and accessible models to mimic in vivo responses. To obtain reliable data, assessing the health of cultured cells prior to any assays is essential. Furthermore, many cell-based assays require quantification of cell growth. Cell health and growth can be determined by quantifying cell viability, proliferation, or apoptosis. Below, we compare some commonly used assays to help you determine which type is suitable for your experimental design.

• Cell viability assays enumerate the ratio of live and dead cells in a population. Cell viability can simply be achieved by staining and counting live or dead cells.  A deeper assessment of cell health can be attained by measuring cell metabolic activity, such as the ability to reduce tetrazolium salts in MTT and WST-1 assays.
• Cell proliferation assays assess dividing cells. Some common assays include BrdU incorporation (BrdU Assays) that can directly measure DNA synthesis,

In this final installment on how to improve your qPCR gene expression profiling (see 6 Tips to Improve qPCR: part 1 and part 2 for previous discussion), we will discuss pipetting tips and the benefits of a separate reverse transcription step in qPCR template preparation.

Tip #5. Good Laboratory Practice (GLP) with pipetting helps to improve qPCR accuracy.

qPCR is a highly sensitive means for gene expression analysis because it amplifies its template exponentially, however, any deviations in each reaction are also amplified. A major source of such deviations is pipetting error. What can you do to prevent these deviations?

A good place to start would be by using a master mix, or a mixture of common reaction reagents such as DNA template, polymerase, qPCR dye and primers, instead of adding each reaction component individually. Using a master mix can significantly lower the frequency of pipetting and reduce pipetting error. To  account for well-to-well variation using an internal control,

After watching displays of astounding athletic prowess in the 2016 Olympics, I was inspired to take a closer look at the science behind exercise training, recovery, and injury with a focus on the importance of blood vessels during exercise.

Let’s start with some basic training: Why are blood vessels important for exercise?

Muscles need oxygen and nutrients to breakdown fats and carbohydrates for energy and the main delivery system to provide these is blood vessels.  Under normal conditions, a delicate balance is kept between quiescence and remodeling in blood vasculature to maintain a baseline level of muscle activity, but that balance is upset with physical stress such as exercising due to an increased demand for energy and the components required to make that energy.  The “angiogenic switch” is a popular term for the point at which blood vessels change from a quiescent state to an active remodeling state, such as in tumorigenesis [1].  Chemical regulation of angiogenesis is well-researched

Despite being essential for any experiment involving DNA replication, people rarely give primers a second thought.  To coincide with the launch of our new qPCR gene reference tool, we’ll be giving them a little more of the praise they deserve.

Primers are the foundation for such grand purposes as diagnosing diseases, tracking epidemics, creating phylogenetic trees, genetic fingerprinting, and cloning a woolly mammoth.  On a smaller but no-less grand scale, these simple 15-30 nucleotide sequences are widely used to assess cell phenotypes, identify signal transduction aberrancies, perform functional gene analyses, and study molecular pathologies.  The most common research application of primers today is probably in performing the polymerase chain reaction (PCR) to amplify or replicate a targeted DNA sequence.  Like the humble primer, the PCR’s advent revolutionized scientific

In this next installment on how to improve your qPCR gene profiling (see 6 Tips to Improve qPCR for previous discussion), we would like to discuss the limitations of melting curve analysis and PCR inhibitors.

Tip #3. Melting curve analysis is NOT sufficient for assessing qPCR product specificity.

In a melting curve analysis, the number of peaks is commonly used to determine the purity of qPCR products, but is this accurate enough? While having only one peak may be a good indicator of product purity, melting curve analysis can produce both false negatives and positives.  Here’s how:

In dye-based qPCR, a DNA-binding dye (e.g., SYBR^® Green) fluoresces when bound to double-stranded DNA (dsDNA, i.e. your qPCR product), and this increase in fluorescence as dsDNA is produced over time is quantified.  In contrast, a melting curve analysis is essentially the opposite measurement.  In a melting curve analysis, temperature is gradually increased to "melt" or dissociate dsDNA products.  As

Gene expression analysis is critical for understanding the transcriptome profiles of primary cells and how they directly influence the cells’ functionality. In addition, the transcriptome profile can affect cell proliferation, differentiation, senescence, or apoptosis. Traditional reporter-gene assays and cDNA microarrays often require either transfection of exogenous material or large quantities of high quality RNA. Primary cells, however, are notoriously difficult to transfect and the efficiency varies greatly between different cell types. In addition, primary cells have a finite lifespan and limited expansion capacity, making it difficult to obtain a high yield of RNA.  To help address these challenges, ScienCell Research Laboratories has developed GeneQuery™ qPCR Array kits for gene expression profiling which we validated and optimized using primary cell cDNA. GeneQuery™ will enable researchers to directly measure protein expression patterns of primary cells, stem cells, tissues,

qPCR array provides a quick, powerful and sensitive approach for gene expression profiling. To help our customers acquire accurate and consistent results, we are happy to share some tips for better qPCR.

Tip #1. Good primer design is the key to a successful qPCR array.

The success of a qPCR array depends on primer specificity and efficiency, so good primer design is extremely important.  The general rules of primer design for traditional PCR also apply for qPCR arrays: 40-60% GC content; 3'-end GC clamps; no repetitive sequence; no self-dimer, cross-dimer, or hairpin formation; and no long stretches of polypurines or polypyrimidines. Furthermore, there are additional aspects to consider when designing primers for qPCR arrays. First, the amplicon length should be around 100-200bp.  If the amplicon length is too short, the PCR product size will be too similar to the primer dimer to distinguish them from one another without sequencing.  If it is too long, the PCR efficiency will decrease

Dye-based qPCR assays, such as SYBR® Green and EvaGreen®, have many advantages over probe-based qPCR assays. Dye-based systems are desirable because they are highly adaptable, cost effective, and time efficient. qPCR specificity, however, is a vital factor to consider when using dye-based qPCR assays. The dyes can bind to any double strand DNA, regardless of whether it is the desired qPCR product, a non-specific amplification, or a primer dimer.

Low specificity is mainly caused by non-specific binding of primers, which includes binding to similar sequences in the DNA template at random locations, binding between primers, and primer self-binding. In addition to careful primer design to exclude undesired primer complimentarity, there are many other ways to improve qPCR specificity. The most effective way to improve specificity is to design primers about 23-26 nucleotides long, with a high annealing temperature (Tm) around 65°C. This is especially important when using genomic DNA or cDNA