Gene Expression Analysis Solutions from Thermo Fisher Scientific
Thermofisher Scientific offers a broad range of solutions for studying gene expression and genomic diversity that fit the varying needs of today’s research laboratories. The ability to quantify gene expression level within a cell tissue or organism can provide valuable information on gene response to biological conditions such as pathogen infection, exposure to chemical compounds, organ or tissue location, cell cycle, differentiation status or disease states. Advanced DNA and RNA sequencing technology has created large pools of data that are being studied to better understand the meaning and consequences of variation in genomic DNA and its transcription products.
Quantitative PCR (qPCR)
Real-time or quantitative PCR (qPCR) is used for a broad range of applications, including quantification of gene expression, measuring RNA interference, biomarker discovery, pathogen detection, and drug target validation. Quantitative PCR can be used to investigate changes in the quantity of particular gene products or a set of gene products.
Thermo Fisher Scientific offers a complete suite of Applied Biosystems™ products for the entire qPCR gene expression analysis workflow—from sample prep to reverse transcription and thermal cycling to data analysis. Real-time PCR is a fast, easy, and affordable technique for studying gene expression analysis and SNP genotyping. Results from microarrays and RNA sequencing are often validated using real-time PCR with Applied Biosystems™ TaqMan® Assays.
Applied Biosystems™ TaqMan™ Assays represent the most trusted and comprehensive collection of qPCR assays available. Over 10 million pre-designed TaqMan™ Assays are available for more than 25 species in various formats: single tube, assay plate (96-well, 384-well), 384-well microfluidic array card and open array plate formats. The TaqMan™ Assays can be used in Gene expression analysis/quantitation, Single Nucleotide Polymorphism (SNP)-based genotyping applications, Copy Number Variation (CNV) analysis, Mutation analysis or microRNA (miRNA) analysis. For complex analysis of multiple genes in a molecular pathways convenient and cost effective solution is the use of Array plates or 384 well microfluidic cards. Beyond the Fixed content TaqMan™ Array plates or cards customers can select their content from the pre-designed assays and create a TaqMan™ flexible content panel and further customisation is possible to add customer designed assays to the Custom TaqMan™ Array plates.
Figure 1.: Applied Biosystems™ TaqMan™ Assay Formats
Applied Biosystems™ TaqMan™ Gene Expression Assays
Applied Biosystems™ TaqMan™ Gene Expression Assays (PCR primer and TaqMan probe sets) are tested and optimized for specificity, reproducibility, linear dynamic range, sensitivity, and efficiency in gene expression analyses. Thermo Fisher Scientific’s TaqMan Assay Search Tool allows users to find the TaqMan™ Gene Expression Assay they need for their research.
How are these assays designed and created?
Most TaqMan™ Gene Expression Assays are designed using transcript sequences from the NCBI Reference Sequence (RefSeq) Project database , which is the most highly curated set of non-redundant transcripts in the public domain. The design pipeline uses sophisticated bioinformatics algorithms to select assays with the highest transcript specificity and optimal thermodynamic and chemical properties. TaqMan™ Gene Expression Assays are evaluated for annotation updates and the latest genome information. Most assays are found to align with multiple transcript accession IDs. This information is displayed in the TaqMan™ Gene Expression Assays online search tool, so that researchers can easily see all the accession IDs with 100% identity to the assay probe and primers, as well as amplicon size, and the assay location on each detected transcript. Each TaqMan™ Gene Expression Assays receives an AssayID in the on-line research tool, making the ordering process more user friendly and convenient. The Assay IDs carry various suffixes (see table 1) with important information about the assay placement in the target. Together with this information the users can choose the Pre-Designed Assay best suited for their experiment.
If the desired assay is not found among the pre-designed TaqMan™ Gene Expression Assays Custom TaqMan™ Assay Designs are available, where customers can submit their primer and probe sequences to create their custom assay ID. During the design process there are two levels of bioinformatics QC available. The Standard Custom TaqMan™ Gene Expression Assays do not offer bioinformatics or QC checks performed on the custom sequences, however if that’s required the Custom Plus TaqMan™ Assays are the right choice. Custom Plus TaqMan™ Assays are designed using the full bioinformatics power of the TaqMan Assay design pipeline, which uses proprietary algorithms that have generated millions of assay designs and is regarded as the benchmark in the industry for qPCR design.
Table 1. Applied Biosystems™ TaqMan™ Assay ID Suffixes
Applied Biosystems™ TaqMan™ Advanced miRNA Assays
Applied Biosystems™ TaqMan® Advanced miRNA Assays enable highly sensitive and specific quantification of mature miRNAs that are ideal for analysis of multiple miRNA targets from a single sample, or low-level RNA samples such as serum and plasma. The Assays detect as few as 60 copies of target input into cDNA synthesis, and are able to quantify mature miRNA from as little as 1 pg of total RNA or 2 μL of plasma. For best performance and improved detection of low-expressing miRNA targets it is recommended to use the TaqMan™ Advanced miRNA cDNA Synthesis Kit. TaqMan® Advanced miRNA Assays detect only the mature miRNA and distinguish between highly homologous targets. TaqMan® Advanced miRNA Assays are also available in variable formats Single tube, Array Plates, Array Cards and open Arrays.
Digital PCR - Detect low-fold changes in gene expression
Real-time PCR is commonly used to detect differential gene expression; however, this approach is generally limited to detecting changes that vary by two-fold or more. For some studies, detection of expression changes less than two-fold may be required. Furthermore, it is often necessary to express differential gene expression with respect to a reference gene, such as a housekeeping gene like actin. Applied Biosystems™ QuantStudio™ 3D Digital PCR System uses molecular counting to provide highly precise and sensitive results. The simple and affordable platform uses a sealed chip technology, providing a streamlined, reliable, and robust method for performing digital PCR. The platform is ideal for applications such as absolute quantification of differential gene expression and rare-allele detection. With the ability to achieve highly precise measurements of ±10% or better, digital PCR is capable of resolving changes of two-fold or less. Like real-time PCR, digital PCR requires the conversion of RNA to cDNA. Since the efficiency of conversion is important to experimental sensitivity, Thermo Fisher Scientific offers the High-Capacity cDNA Reverse Transcription Kit, which seamlessly integrates into your digital PCR gene expression workflow.
QuantiGene™ RNA Assays for Gene Expression Profiling
Quantitative PCR has inherent inefficiencies due to RNA extraction method efficiency/bias and variability introduced by human error. As an alternative Thermo Fisher Scientific offers alternative approach using Invitrogen™ QuantiGene™ RNA assays, that provide a more robust and accurate measurement compared to other gene expression quantitation methods.
Invitrogen™ QuantiGene™ RNA assays are hybridization-based assays that utilize a branched DNA technology for signal amplification for the direct quantitation of gene expression transcripts. With this method it is possible to quantitatively measure RNA directly from cell or tissue homogenates and even directly from blood without RNA purification or enzymatic manipulation.
The Invitrogen™ QuantiGene™ RNA assays are available in two formats:
The QuantiGene™ Singleplex Assay detecting single transcript is performed in a 96-well plate (or 384 well plate Singleplex HT Assay) and signal is detected using a standard luminometer.
The QuantiGene™ Plex Assay utilizes xMAP Luminex beads for multiplexing of 3 to 80 RNA targets followed by branched DNA-based signal amplification. Choose from a variety of preassembled panels or custom design your own plex using our design tool. The assay is performed in a 96-well plate and signal is detected using a Luminex instrument.
Figure 2.: QuantiGene™ vs qPCR workflow
Ion GeneStudio S5 Series and Applied Biosystems™ Clariom™ assays - Thermo Fisher Scientific Solutions for Transcriptome analysis
For multiple targets and high throughput Gene expression analysis Thermo Fisher Scientific also offers Next Generation Sequencing and Microarray solutions.
Applied Biosystems™ Clariom™ assays can generate expression profiles from as little as 100 pg of total RNA and as few as 10 cells. RNA can be analysed from a wide variety of sample types, including cells, whole blood, and fresh/fresh-frozen or formalin-fixed, paraffin-embedded (FFPE) tissues. The technique doesn’t require globin or ribosomal RNA removal. The free Transcriptome Analysis Console (TAC) software performs simple and fast transcriptome analysis. Two assay types are available:
Clariom D assays perform comprehensive, detailed analysis of coding and long noncoding genes, exons, and splice variants
Clariom S assays rapidly generate robust expression profiles from all well-annotated genes.
Table 2. Applied Biosystems™ Clariom™ assays
Using Ion Torrent™ NGS technology users can survey differential gene expression profiles and discover novel biomarkers. The Ion GeneStudio S5™ series offer scalable throughput for whole transcriptome RNA-seq, targeted RNA sequencing and small RNA sequencing. With Ion AmpliSeq™ technology it is sufficient to use as little as 1 ng of input RNA to detect multiple RNA targets in parallel within the sample.
In summary, depending on throughput, sample and instrument availability customers can use different approaches to study Gene Expression and cell response to environmental conditions.
Which gene expression technologies are right for you?
Gyongyi Lukacs, PhD
Field Application Specialist