Gene Expression Analysis using Real Time PCR
Gene expression profiling is a powerful tool that measures gene activities (expressions) at the given cell for a certain time frame, to process thousands of cells at the same time, and generate a global map of cellular function can be generated. The quantification strategy is an important factor for detecting of mRNA gene expression level. Quantification of mRNA transcription can be measured by absolute or relative quantitative Real-time PCR. Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput.
Relative quantification determines the changes in steady-state mRNA levels of a gene relative to the levels of an internal control RNA. This reference is usually a housekeeping (HK) gene that is either co-amplified in the same tube or amplified in a separate tube. Therefore, relative quantification does not require standards with known concentrations and all samples are expressed as an n-fold difference relative to the HK gene and the number of target gene copies are normalized to the HK gene.
Absolute quantification provides a more accurate and reliable, albeit more labour-intensive method for the quantification of nucleic acids. CTs obtained from an unknown sample are compared to CTs generated from a series of samples of known concentration or copy number. Results can be expressed as copy number per unit mass, e.g. μg total RNA. The expression of a target nucleic acid is usually compared across many samples, often from different individuals and sometimes from different tissues.
Advanced Level: Starts from RNA isolation to data analysis using either the standard curve method or relative expression (ddCt).
Intermediate Level: Starts from RNA isolation to the qPCR run without performing data analysis.
Basic Level: Once you submit extracted RNA, we perform a quality check of your samples using Nanodrop, and gel electrophoresis. When the samples pass QC, we notify you that we are ready to proceed with the qPCR run and data analysis.
Requirements: Customer provides the following
Fresh tissue/blood/plant samples, or extracted RNA (Vol. = >30μl ; Conc. = >100ng/μl) .
Specific primers to detect the gene/fragment of interest. Alternatively, customer may purchase the primers available from us.
Sample expressing the target gene as the positive control.
Once the lab work and data analysis are complete, we send you a service report including:
Row data ( ct values ) .
Data Analysis ( Standard Curve / Fold Change ) .
Complete methodology part .
Turnaround time is about 1-3 weeks. Actual times vary based on how many projects we have.
Why Gene Expression Service in CEB
Because our services provide the expertise for the measurement of mRNA and offer an accurate and sensitive method for gene-expression analysis, accelerating the understanding of changes in gene expression across different tissues, blood types, plants, and individual cells. We are equipped to perform both SYBR and Probe-based qPCR assays.