Gene Expression Analysis

Command absolute quantification of RNAs and gene expression analysis for low copy number templates by using reverse transcription.

Cells containing your RNA of interest are lysed followed by RNA extraction to isolate RNA for gene expression profiling. Careful sample handling, as well as RNAse inactivation and RNAse free solutions and consumables, is essential to ensure RNA integrity.

Reverse transcription is a process by which enzymes catalyze the transcription of RNA into a complementary DNA (cDNA) using reverse transcriptase. The cDNA in turn is the template for PCR amplification, followed by detection.

A successful qPCR assay requires efficient and specific amplification of the RNA transcript. The choice of target sequence, plex level, and primer design is significant, as they can affect amplification efficiency and specificity and thus the accuracy of qPCR assays.

In RT-qPCR, the amplification reaction is set up with PCR reagents, primers and probes in a master mix, and run using real time PCR instruments. The target specific fluorescence from the amplification reaction is monitored in real time and is proportional to the amount of amplified product.

Quantifying the absolute amount of a target sequence or comparing relative amounts of a target sequence between samples uses the Cq value and is calculated by your cycler's proprietary software. This value determines the PCR cycle number at which your sample’s reaction curve intersects the threshold line and provides a real signal. Real-time PCR has a reaction curve for each sample and therefore many Cq values.

Gene expression by RNA sequencing provides a holistic view of the transcriptome, making it ideal for transcript discovery.

Implementing a fit-for-purpose combination of gene expression analysis techniques can yield better outcomes, save you time and money, and help you make discoveries faster.

Learn how to capture more of the transcriptome during gene expression profiling from FFPE RNA samples using the SEQuoia Complete workflow.

Our Droplet Digital PCR Master Class video series provides an overview of the fundamentals of digital PCR.