Frequently asked questions about real-time qPCR

Q 01: Should I choose probe or SYBR Green chemistry for my experiments?
When you are optimizing a new experiment and you need to test many primer pairs, it is usually best to start with SYBR Green. SYBR Green enables you to find the most specific primers without the need to design and purchase several probes. SYBR Green also allows melting curve analysis, which can reveal undesirable primer-dimers or other nonspecific PCR products. Probe chemistries are better when you require more specificity in detection (e.g. discrimination between closely related species). Probes are also needed for multiplex reactions that allow you to detect several targets at once.

Q 02: Why is my amplification efficiency over 100 %? Why is my standard curve not a straight line?
PCR efficiency depends on the method used to determine it. If the efficiency is calculated based on a standard curve, the slope of the curve may be skewed at one end or the other by the late detection of a large concentration of product due to inhibitors, or by the early detection of nonspecific PCR products or primer-dimers. The skewed slope directly affects the calculated efficiency. An efficiency over 100 % can also be the result of an inaccurate standard dilution series. This can be avoided by careful use of calibrated pipettors when preparing standard dilutions. The qPCR efficiency can be calculated from the slope of the standard curve with the qPCR efficiency calculator at Finnzymes’ website.

Q 03: Why is the amplicon length so limited in qPCR?
The amplification efficiency tends to decrease as the amplicon length increases. Short amplicons are more likely to be completely denatured during the denaturing step, making the annealing of primers and probes more efficient. Also, shorter amplicons are copied more rapidly during the extension step.

Q 04: Should/Can I use some additives to improve my qPCR results?
Finnzymes’ DyNAmo qPCR kits are already optimized to give the best possible results with the majority of amplicons. Generally the best way to further improve qPCR results is to redesign primers or adjust the primer and probe concentrations.

Q 05: How can I analyze the quality of my RNA?
A rough estimate of RNA purity can be obtained from spectrophotometric data. The ratio of A260/A280 nm should be close to 2.0. Impurities such as proteins can change this ratio. The pH has also an effect and thus RNA purity measurements should be carried out in buffered solutions. A more thorough quality assessment can be performed with electrophoresis. The ratio of peaks from ribosomal RNA in an electropherogram can be used to determine the degree of RNA degradation. A 2:1 ratio of 28S to 18S is considered good. Some electrophoresis systems use also other criteria to determine a quality value for RNA (e.g. RNA Integrity Number, RIN, from the Agilent Bioanalyzer system). However, the RIN value of a sample does not necessarily correlate with its success in qRT-PCR. In addition to previously mentioned, a 3’:5’ assay can be used to assess mRNA degradation by determining the proportion of full-length cDNA transcribed (reviewed in Nolan et al. (2006) Nat. Protoc. 1:1559-1582).

Q 06: Can I pipet a qPCR plate in advance and store it for later use?
When using a hot start polymerase it is not critical to start reactions immediately after setup. Yet, storage at room temperatures is not recommended for longer than a few hours. A prepipetted plate can be stored at +4°C up to a day. Reactions can be stored even longer periods at -20°C. However, the storage conditions can have some effect on performance and thus it is recommended to treat all reactions in one experiment similarly.

Q 07: Why do I get several peaks on my SYBR Green melting curve even though the amplification efficiency is good?
There can be several reasons for abnormal melting curve data. Nonspecific amplification does not necessarily have an impact on the amplification efficiency and even if it does, the effect can be difficult to see. Additional peaks may also form as a result of primers binding to a closely related gene. It is important to check primers for specificity using a BLAST search.

Q 08: What is ROX used for?
ROX is a passive reference dye that can be used for normalizing against non-PCR related fluorescence signal variation. ROX does not take part in the PCR reaction and its fluorescence remains constant during the PCR reaction.

Q 09:How much ROX is needed for each instrument? Why are the ROX concentrations different?
The amount of the ROX passive reference dye needed can vary depending on the instrument optics. More ROX dye may be needed with real-time PCR instruments which use an argon laser as the light source or which have excitation filters not optimal for ROX dye.

Q 10: How should I compare data from different runs?
For different runs to be comparable, the same PCR reagents, reaction setup and analysis settings must be used. Also, an inter-run calibrator needs to be included in the runs, and it’s preferable to have reference standards in all the runs.

Q 11: What is a true positive?
It is crucial that qPCR assays are validated thoroughly in order to be confident that the results are meaningful. Results are often questionable if samples are detected in very late cycles where the calculated copy numbers would be below zero.

Q 12: How many reference genes do I need?
In relative quantitation, reference genes are used to normalize against variation in sample quality and quantity. The number of reference genes needed depends on the assay, the accuracy needed and the references chosen. Several reference gene candidates should be tested to find the most suitable combination. There are many software tools available for validation of reference genes.

Q 13: At what point in the experimental process should I split the samples into replicates (extraction, RT, or qPCR)?
It is better to have true biological replicates and replicates already in reverse transcription step. It is important to use replicates in the steps where the most variation is introduced.

Q 14: When should I use carrier DNA or RNA?
Diluted nucleic acids can bind to walls of reaction vessels and pipette tips. This may cause problems especially if a very low amount of RNA or DNA template is available per reaction. Carrier DNA/RNA can saturate surfaces and prevent losses of the target nucleic acid. Carrier can also normalize the total nucleic acid concentration in the reaction so that all samples are amplified in the same conditions. Furthermore, carrier can protect low concentrations of target RNA from degradation by RNases. For example, genomic DNA can be used as carrier with DNA samples. With RNA samples the carrier can be yeast tRNA. It is important that the carrier does not contain the specific target sequences.

Q 15: Is it preferable to use elevated temperature in the RT reaction?
When template RNA has stable secondary structures, it can be beneficial to use elevated temperatures in the reverse transcription reaction. Some RT enzymes are active even at temperatures up to 70°C. However, higher temperatures induce the degradation of RNA, and this is why RT temperatures over 42°C are rarely used.

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