Incorporate qPCR into your Cell Based Assay

At Visikol, we offer our Clients an array of 2D and 3D cell culture assays with a wide range of imaging-based end points that allow our Clients to answer complex research questions using quantitative image analysis. To continually improve our services, we are always looking for new ways to provide even more insights to our Clients and to expand beyond our best-in-class imaging-based end points. That is why we are proud to announce the incorporation of qPCR to the suite of services we offer to our Clients such that we can now provide multiple end point read outs from the same assay.

qPCR is a well validated, widely used method to quantify a host of relevant biological information. It may be applied to cell-based models to quantify gene expression, gene abnormalities, viral infection, mycobacterial infection, etc. For example, Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) is a great tool for observing changes in gene expression for cell-based assays. This method reverses the process of transcription to convert the RNA from a sample into complementary DNA (cDNA). RT-qPCR is a great tool to look at differences in how cell populations express their genes in response to stimuli.

Polymerase Chain Reaction (PCR) amplifies small amounts of DNA sequences in an exponential fashion. It relies on thermal cycling or exposing the sample and reactants to repeated cycles of heating and cooling to denature, anneal, and elongate the target DNA. With each thermo cycle the number of replicate DNA strands doubles; this allows for the rapid, exponential reproduction of the DNA sequence of interest.

The PCR reaction relies on two main components: primers and DNA polymerase. Primers are short, complementary (to the gene of interest), single stranded DNA fragments that catalyze the replication of DNA. Two primers are needed per gene of interest, one that compliments the begging of the sequence (forward primer) and one that compliments the end of the sequence (reverse primer). DNA polymerase is a heat-resistant enzyme that carries out the replication of the full DNA strand of interest.

As the DNA of interest replicates, it is quantified by a fluorescent dye. There are two main types of dyes used in qPCR: Intercalating Dye Assay and 5’ Nuclease Assay. Intercalating dyes (ie. SYBR Green, Cyto, EvaGreen) are double-stranded DNA binding dyes. This class of dyes have low background fluorescence that increases upon the formation of double stranded DNA. This is due to the dye’s ability to fluoresce when bound to double stranded DNA. As the target DNA replicates, the signal intensity from the dye increases proportionally to the number of replicates formed. Intercalating dye qPCR is a cost-effective, rapid, reproducible way to quantify genes of interest. Two disadvantages to intercalating dye based-methods are: that they detect any replicated DNA product, regardless if they are in fact the DNA sequence of interest; and primers may not be multiplexed, meaning only one target DNA sequence may be probed at a time.

5’ Nuclease dyes (i.e. TaqMan or PrimeTime) incorporate a probe sequence, in addition to the forward and reverse primers. The probe’s sequence is complimentary to the gene sequence of interest. This probe sequence has a fluorescent marker attached to one end and a fluorescence quenching molecule attached to the other. As the DNA polymerase elongates, the second strand of the replicated DNA cleaves the fluorescent marker from the probe allowing it to fluoresce. Therefore, the intensity of fluorescent signal is directly proportional to the number of replicated DNA strands. Also, due to the nature of 5’ Nuclease dyes, target sequences may be multiplexed, meaning one or more targets may be probed for at a time. Multiplexing is possible as different probes may fluoresce at different wavelengths and are highly specific to their target gene sequence. While 5’ Nuclease dyes do account for some of the intercalating dye’s disadvantages, they are more expensive, require more rigorous optimization, and may interact with one another in unforeseen ways when multiplexed.

If your company is in search of a novel approach to speed up your drug discovery pipeline with biologically relevant models, Visikol may have what you are looking for. At Visikol, we have developed several standardized and validated 3D cell culture models utilizing cancer cell lines, primary tumor cells, as well as liver models. These models provide access to assays to evaluate cancer drug efficacy, drug induced liver injury (DILI), as well as to model various liver disease pathologies such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD).

2020-06-25T14:21:44-05:00
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