Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

In this video article, real time PCR is demonstrated using Solaris QPCR assays. Solaris QPCR assays are gene specific probe and primer repairs designed to detect all known splice variants of a given gene. Under universal thermocycling conditions, the Solaris probe has a minor groove binder, moiety and FA reporter on the five prime end and an eclipsed dark quencher at the three prime end.

During the denaturation step of real-time PCR, the probe remains in solution and the FAM reporter fluorescence is quenched. During the an kneeling in extension phases, the primers in probe bind to the target sequence and the quencher is separated from fam. FAM then gives a strong fluorescent signal when the probe dissociates fluorescence is again quenched.

Real-time PCR results are obtained that demonstrate the sensitivity, specificity, and reproducibility of this efficient assay. Hi, I’m James Covino from the Thermo Fisher Scientific Research and Development Team in Lafayette, Colorado. Today we’ll show you a procedure for setting up a standard QPCR reaction using Lars QPCR gene expression reagents.

We use this procedure in our laboratory to study the knockdown of targeted genes using RNA interference technologies. So let’s get started. The Solaris QPCR assay STEM is defined by its unique algorithm that utilizes MGB and super base technologies to provide a single convenient assay for 98%of human and mouse annotated genes.

Typically, DNA probe and primer design is restricted by the melting temperature of the probe, which is determined by the number and type of bases. Longer sequences and GC rich sequences have higher melting temperatures. The minor groove binding moiety used in Solaris probes fits into the minor groove of the DNA helix and stabilizes the probe target sequence interaction.

This increases the melting temperature, allowing shorter probes to be used. The Solaris algorithm also selectively incorporates modified nucleotides called super bases to improve the stability of at bonds and eliminate GG self association in guine rich sequences. This allows for the use of target sequences that would otherwise be avoided.

The increased sequence flexibility conferred by the use of these technologies enables the design of primer probe sets that recognize all known splice variants of a single gene. Note that whenever possible exon junction spanning regions have been incorporated in the target site to increase specificity by avoiding the amplification of contaminant genomic DNA, the resulting primer probe sets blast analyze using three different databases, genomic transcript, and pseudogene to ensure specificity. Furthermore, the Solaris primer probe sets have been completely optimized such that the cycling conditions for all QPCR assays are identical, which allows for the expression of various transcripts to be assessed in single PCR runs.

To begin planning the qPCR R experiment, identify the reagents needed to perform qPCR for the gene of interest using the genius product search, which can be found at thermo.com/solaris. Click in the search inquiry box and choose the appropriate target organism. The genius product search can process many different gene identifiers.Here.

Enter one of the suggested search terms to the gene of interest, then click on search, identify the gene of interest and click on select on the left-hand side. This will open a window that shows the different reagents available For the gene of interest. Click on the QPCR detection tab under the Solaris gene expression assay.

Heading one optimally designed Solaris probe primer assay is displayed. This one assay will detect all known splice variants of the gene of interest. Once the primer appropriate agent has been identified, find the QPCR cycler that will be used on the menu and identify the compatible master mix, either the master mix with a separate rocks passive reference die vial, the rocks mix, or the low rocks mix.

Here we’ll be using the Roche light cycler four 80, so the master mix plus rock vial will be used. It is also important to ensure that an appropriate reference gene is included in the study here. B two M will be used once all of the Solaris QP CR reagents are received.

They should be stored at minus 20 degrees Celsius until they’re used. The reagents are stable for at least 12 months. Repeated freeze thawing should be avoided.

Prepare CDNA for QPCR by performing reverse transcription reactions on sample RNA. Once the CDNA has been prepared, thaw the Solaris assay and master mix on ice. The master mix contains all components for quantitative PCR except the gene specific assay and template components include thermostat, DNA polymerase dn, TPS rocks.

If the QPCR instrument requires rocks and a proprietary reaction buffer containing an inner blue dye, also have on hand PCR grade water and QPCR plates. Once the solutions have thawed mixed by flicking, then briefly spin the solutions down to prevent loss of reagent. Next, in a 15 milliliter tube, prepare one x Solaris QPCR assay and master mix.

By combining for the desired number of samples to be run on a 384, well plate five microliters of two x master mix 0.5 microliter, 20 X Solaris assay and PCR grade water so that the final volume in each well will be 10 microliters. After the CDNA is added, be sure to prepare enough for three replicates of each sample, including a no template control NTC and A.No reverse transcriptase control, no RT to check for contaminated reagents or contaminating genomic DNA respectively. Next, transfer the mix into a sterile reservoir.

Then using a multi-channel pipetter transfer the appropriate volume of one x master mix to the appropriate wells of the PCR plate. If white plates are used, the inner blue dye included in the master mix will aid in tracking pipetting progress. Multiple CDNA libraries and reference genes can be assayed on the same plate since all cycling conditions are identical.

Once the one x master mix has been added to the appropriate wells, add diluted CD NA template to each well so that the final volume is 10 microliters pipette up and down. To mix. Seal the plate with either an adhesive seal or a heat seal as shown here.

Place the plate in a centrifuge and spin down to bring the reagents to the bottom of the well and remove any bubbles which will interfere with fluorescence readings program. The thermocycler, the hot start polymerase in the Solaris QPCR master mix must be activated at 95 degrees Celsius for 15 minutes, one cycle, then 40 cycles of 95 degrees Celsius denaturation for 15 seconds, and an kneeling an extension at 60 degrees Celsius for 60 seconds. This protocol is the same for all Soliris QPCR assays, regardless of the gene being amplified.

Finally, place the plate in the QPCR instrument and start the program. This figure demonstrates the high performance of Solaris assays here, 10 tenfold dilutions of synthetic DNA or CDNA were amplified using the Solaris QPCR gene expression assays for CDC 20 and F two RL one, the log scale amplification curves and standard curves for 10. Log 10 dilutions demonstrate the high performance of the assay even at low input concentrations.

The performance of each assay is measured by the efficiency are squared value dynamic range out of 10, log 10 dilutions and the lower limit of detection. The data shown here demonstrate the reproducibility of Solaris assays. Here, aliquots of CD NA were amplified in two geographically separated laboratories using Solaris QPCR gene expression assays and master mix.

The same expression levels were calculated for two gene targets in both laboratories. We’ve just shown you how to set up a standard QPCR reaction using Solaris QPCR gene expression reagents. When performing this experiment, it’s important to remember to set up master mixes with the correct final concentration of reagents and include appropriate biological and or technical replicates.

In addition, it’s imperative to set up the QPCR instrument with the correct thermocycling conditions for Solaris. So that’s it. Thanks for watching and good luck with your experiments.