When do you use Rox in your real-time PCR?

ROX is a fluorescent molecule that the real-time PCR system can detect when its present in the reaction. It’s used as a Passive reference dye for normalization of fluorescence signal across all of the PCR samples of the PCR thermo cycler including a baseline.

Rox is required when there is uneven illumination, sample variation and difference in quantity or condensation.

Its shadowing the reporter as a constant fluorescent and results in a higher precision of well data.

Adding ROX depends on the Real-time PCR instrument you’re using. Companies such as for example Bioline offer an easy selection tool on their website, informing you about the exact requirements for your instrument.

What is the difference between SensiMix and SensiFast?

The SensiFAST and Sensimix kits has been developed for fast, highly-sensitive and reproducible qPCR and has been validated on commonly-used real-time PCR instruments and is designed for superior sensitivity and specificity with probe-detection technology, including TaqMan®, molecular beacon and Scorpions® probes. Consistently accurate detection of DNA and RNA targets from a broad range of sample types and Excellent efficiency for improved multiplexing

Difference :

Sensifast includes The use of an antibody-mediated hot-start DNA polymerase which minimizes amplification from primer-dimers, thereby improving assay specificity and sensitivity. Sensimix includes a Covalently-modified hot-start promotes highly-specific amplification

Sensifast is the latest version and improved Reproducible. Faster accurate results in as little as 30 minutes.

When do I choose a 1-step kit and when do I choose a 2-step kit?

1 step kit involves including the reverse transcriptase step in the same tube as the PCR reaction and the 2-step kit involves creating cDNA first.

1 step:
Using gene specific primers, one-step real-time RT-PCR such as the One-Step kits offer a quick and simple method to detect mRNA and so are useful when analyzing a few genes over a large number of samples as less pipetting and sample manipulation reduces variation and potential contamination. However reaction conditions needed to support both the RT and PCR may not be optimal for either reaction and it is not possible to archive the cDNA produced during the reverse transcription reaction.

This method is quick to set up and makes processing multiple RNA samples easy (especially when using liquid handling robotics), when you are amplifying only a few genes of interest. It is therefore ideal for high throughput screening laboratories where only a few assays are run repeatedly, using well-established reaction conditions, with the added advantage that multiplex PCR of the gene of interest and control genes can be done in single well.

2 step:
Two-step real-time RT-PCR, offers a truly accurate determination of mRNA and is useful when analyzing a large number of transcripts over a few samples. SensiFAST™ kits have flexibility in the priming strategy, allowing for oligo-dT, random primers or gene specific primers and are generally more sensitive than one-step as the RT and PCR occur separately and can be optimized individually. Also, the cDNA produced is more stable than the initial RNA sample and can be more easily archived for future use.

With two-step real-time PCR, the use of several tubes means that it is more time consuming and less adaptable to liquid handling robotics and so more difficult to adopt for high throughput screening assays. The use of several tubes and pipetting steps also exposes the reaction to a greater risk of DNA contamination

  • Advantages
    – Accurate representation of target copy number
    – Simple and rapid Fewer pipetting steps (reducing possible errors and  contamination)
    – Best option for high-throughput screening
    – Best method when only a few assays are run repeatedly
    – Multiplex PCR of gene of interest and control can be done in single well, from same RNA sample

  • Disadvantages
    – Usually less sensitive as it is impossible to optimize the two reactions separately
    – Difficult to troubleshoot RT step
    – No stock of cDNA

Before choosing you should consider the best one for your application, these include the ease of use and cost of reaction to the resulting yield and sequence representation.

Why should I use a Hot Start polymerase?

The main reason to us a hot start Polymerase (used within a process also known as a ‘Hot Start PCR’) in your PCR reaction is to avoid unspecific amplification. Most DNA polymerases work best at a temperature between 68 and 72°C. In some cases, an enzyme can become slightly active below these temperatures and this will cause unspecific binding, leading to unspecific amplification. A hot start PCR will reduce the nonspecific amplification significantly.

What is a Hot Start polymerase? Technically it is a standard PCR polymerase, but a Hot Start Polymerase is inhibited in its functionality by a structural change to the enzyme. These changes can include antibody interaction, aptamer technology or chemical modification. Typically a Hot Start polymerase needs to be activated by incubating the enzyme at 95°C for a longer period of time to remove the polymerase inhibitor.

Last but not least, when using a hot start polymerase, it provides the advantage of performing the PCR reaction setup at room temperature. Usage of a Hot Start polymerase can therefore be advised when performing high-throughput experiments using liquid handlers or experiments demanding high specificity.

So why not always use a hot start polymerase? Well, it has a disadvantage as well.. The re-activation time during the denaturation stage is increased for activation of the enzyme. This increased heating time could damage your DNA. Studies have also shown that using a hot start PCR can cause issues when amplifying long strands of DNA.

What is the difference between PCR and real-time PCR?

PCR is technically an end-point reaction, it allows for the amplification of a specific DNA segment, based upon the PCR primer annealing sites. In a standard PCR the DNA template is amplified by repeating 3 steps, denaturation, annealing and elongation. After thermocycling the result of a PCR reaction can be visualized using for example agarose gel analysis, capillary electrophoresis, sanger sequencing, etc.

RT-PCR describes a form of PCR allowing the use of RNA as a template. The RT in this case means Reverse Transcription. RNA is in the first step reverse transcribed into complementary DNA (cDNA). In the second step, the single stranded cDNA is completed and amplified into a double stranded DNA product.

qPCR or quantitative PCR will amplify a specific DNA template segment based upon the PCR primers as well. However, it will also allow measurement of the DNA template concentrations. The amplification of the DNA can be measured throughout each amplification cycle, due to the presence of an DNA binding dye, for example SYBR® Green. SYBR® binds only to double stranded DNA and once bound changes its structure releasing a fluorescent signal. Since each PCR cycle accumulates the amount of double stranded PCR product in the reaction vessel, the signal will increase with each cycle parallel with the DNA concentration.
Next to the DNA sample(s), a series of DNA standards will be amplified as well. These standard will help forming a calibration curve, which enables scientist to plot and determine the concentrations of their DNA samples.
qPCR is sometimes also referred to as Real-Time PCR, as it allows scientists to follow the amplification of the DNA template live (real time) throughout the PCR cycles.

RT-qPCR is similar to qPCR, except it starts with RNA as a template. The RNA is reverse transcribed to cDNA and then the cDNA is completed and amplified into a double stranded DNA product in the presence of a fluorescence dye, such as SYBR® Green.

What is PCR?

PCR (polymerase chain reaction) is a technique to make copies of a DNA segment. It allows scientists to create identical copies of a DNA template, to reach a detectable level or for use in other applications as for example NGS or Sanger sequencing.
The process requires a thermocycler, a DNA polymerase, Nucleotides, Primers and of course your DNA template. Once the ingredients for the PCR have been mixed together, the amplification of the template can start using a thermocycler. The thermocycler will perform and repeat a cycle of different temperatures, by heating and cooling the PCR mixture rapidly, facilitating the amplification of the template.

There are three main stages during each PCR cycle:

Denaturation: The double stranded DNA is heated to separate it into two single strands

Annealing: The temperature is lowered to enable the DNA primers to attach (anneal) to the DNA template

Extending/Elongation: The temperature is raised to the optimal working temperature of the PCR enzyme, allowing the enzyme to create and the new strand of DNA, using the Nucletides to build the strand.

After each PCR cycle, the number of DNA strands are doubled.

The exact temperature and time of each stage depend on the primer sequences, DNA fragment lenght and DNA Polymerase being used. In general the temperature of the denaturing stage is 94-95°C, annealing stage is between 50-65°C and extending stage is around 72°C.