Real-time PCR (also known as qPCR) and real-time reverse transcription PCR (also known as RT-qPCR or real time RT-PCR) are widely used for applications ranging from genotyping, gene expression analysis, miRNA and non-coding RNA analysis, and many other purposes. The effective use of qPCR and RT-qPCR requires robust reagents and an understanding of experimental design and data analysis methods.
Answers to frequently asked questions about qPCR and RT-qPCR are presented here. For additional information, refer to the technical literature and web pages for specific Takara Bio products for qPCR and RT-qPCR.
How do you decide between one-step and two-step RT-qPCR?
|One-step RT-qPCR||Two-step RT-qPCR|
|Procedure overview:||Uses gene-specific primers for reverse transcription and allows highly sensitive detection of a specific gene.||
Allows for the preparation of total cDNA by reverse transcription using universal primers that recognize all mRNA molecules, such as random hexamers or oligo-dT primers. The resulting cDNA can be used for the detection of a variety of transcripts.
|When there is an overabundance of total RNA in the reaction:||
Can provide highly efficient amplification even in the presence of large amounts of total RNA.
Using random 6-mers for reverse transcription may result in poor reaction efficiency due to insufficient primer availability.
In this situation, we recommend using oligo-dT primers for two-step RT-qPCR rather than random 6-mers. The use of oligo-dT primers may improve efficiency and provide similar results as compared to one-step RT-qPCR.
Note: When amplifying cDNA produced by oligo-dT primed RT, the PCR amplicon should be located near the 3' end of the expected cDNA.
Analyzing a single gene.
|Analyzing the expression of a large number of genes.|
For high-throughput analyses of many samples or precise detection of rare transcripts, a one-step RT-qPCR protocol is recommended. An additional advantage of one-step RT-PCR is the existence of single-tube workflows, avoiding the need to add additional reagents halfway through the procedure and thus lowering the risk of contamination.
For more information, view our BioView blog post "One-step vs. two-step RT-qPCR—tips for choosing the right protocol".
Which is a better starting sample for preparing RT-qPCR calibration curves, RNA or cDNA?
Calibration curves for RT-qPCR may be prepared by either of the following methods:
- Serial dilution of RNA, followed by reverse transcription and real-time PCR
- Serial dilution of cDNA (obtained by reverse transcription reaction), followed by real-time PCR
Since the two methods evaluate different parameters, it is important to choose a method appropriate for the experimental system being used. Calibration curves prepared from diluted RNA samples reflect differences in not only PCR amplification efficiency, but also differences in reverse transcription efficiency, which is dependent on the amount of RNA. PCR amplification efficiencies determined from such calibration curves may potentially differ from the actual efficiency.
For Absolute Quantification, the reverse transcription efficiency is critical. Therefore, use serially diluted RNA to prepare calibration curves (cDNA dilution is unsuitable).
For Relative Quantification, differences in reverse transcription efficiency can be corrected by assaying a reference gene, such as a housekeeping gene. The use of serially diluted cDNA is recommended for preparing calibration curves.
How can amplification of genomic DNA in total RNA samples be avoided?
To avoid amplification of genomic DNA in total RNA samples:
- Design primers that avoid genomic amplification: select a large intron region and design forward and reverse primers in exons upstream and downstream of the intron. With this strategy, genomic amplifications cannot occur for large introns. When introns are small, genomic amplification can be differentiated based on differences in melting temperature as the result of different amplification sizes (melt curve).
- For genes that lack introns or when the genomic structure is unknown, treat the total RNA with DNase I to remove genomic DNA.
- Use the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Cat.# RR047A). The gDNA Eraser in this kit removes genomic DNA in just 2 minutes, followed by a 15-minute reverse transcription reaction. The resulting cDNA can be amplified using a Takara Bio qPCR premix.
Target gene expression levels are often normalized to the expression of a “housekeeping gene” (reference gene) to correct for differences in the amount of input RNA and variations in reaction efficiency. How do I select a suitable housekeeping gene?
There is no single, universally appropriate housekeeping gene suitable for accurate normalization in every experimental condition, as it is important to select housekeeping genes that do not vary in expression levels in the experimental system used. Common housekeeping genes that have been used in the past include GAPDH and β-actin; however, reports in recent years indicate that expression of these genes may also vary, depending on the sample type and/or experimental conditions.
Using multiple housekeeping genes for normalization is currently the most reliable approach. In this strategy, the expression of multiple housekeeping genes is assayed, and the genes that show the lowest level of variation are selected for use. Software has been developed for selecting the optimum genes for correction (e.g., geNorm and BestKeeper). Inferences may also be made from microarray or RNA-seq expression profiling data, if available.
For additional information refer to: J. Vandesompele, et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3(7):RESEARCH0034.1–11.
qPCR experimental design and data analysis
What standard samples are recommended for preparing calibration curves?
Suitable standard samples are those that approximate the actual sample as closely as possible. For gene expression analysis studies, use cDNA prepared from samples collected under conditions in which the target gene is known to be expressed. For genomic analysis studies, use genomic DNA.
Artificial standard samples (e.g., plasmid DNA) are not recommended. Even when the sequence of the amplicon is identical, any substantial difference in the template composition may result in variable PCR amplification efficiencies (e.g., genomic DNA vs. plasmid DNA).
What method(s) should be used for analyzing qPCR amplification products?
When using a validated primer, previous melt curve analysis (Tm values) can serve as a reference. If the Tm value is the same as observed in the past, it is reasonable to assume that the PCR product is the same as that obtained in the past.
Keep in mind, however, that although identical PCR products will show the same Tm value, obtaining identical Tm values alone does not necessarily confirm that the PCR products are identical. An independent confirmation method should be used; when using a primer for the first time, please perform electrophoresis to confirm that the amplification product of the real-time PCR is the intended size.
What is the appropriate number of replicates (n)?
The necessary number of sample replicates (n) varies depending on the experimental system. In principle, when the experimental error is expected to be relatively large, use a larger number of samples.
- When profiling gene expression by RT-qPCR, it is useful to prepare multiple biological replicates and perform multiple RNA extractions in order to ascertain the degree of biological variability.
- With respect to qPCR, greater variability is expected when using a low level of template because the number of cycles required for detection will be high. In such cases, use a larger number of replicates.
What is the sensitivity of qPCR?
The sensitivity of qPCR varies depending on experimental conditions such as the reagents and primers used. Appropriately designed systems have demonstrated the ability to detect as few as ~10 copies of template.
I understand that there are two ways to determine the Ct value. What are they?
The Ct value can be determined by two different methods:
- The crossing point method defines the Ct value as the crossing point between the amplification curve and the threshold line.
- The second derivative maximum method defines the Ct value as the point of the maximum of the second derivative of amplification curve (second differential curve).
The latter method allows highly precise analyses, since the Ct value is fixed by setting a threshold and is not subject to the effect of instrument-specific detection variability.
Please note that some instrument systems do not allow one to choose between the crossing point method and the second derivative maximum method.
What are the differences between absolute and relative quantification?
- Absolute quantification determines the absolute number (i.e., number of copies) of a target using a standard sample that has a known number of copies.
- Relative quantifications allow a relative comparison between samples.
Typically, using relative quantification, a target gene to be quantified and a reference gene (e.g., a housekeeping gene) are simultaneously assayed for the purpose of correction (normalization). Relative quantification provides the difference in expression level in the unknown sample compared with the control sample.
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