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Technical Appendix, qPCR and qRT-PCR
qPCR
Template preparation and quality
Quantitation
PCR primers
MgCl2 optimization
UNG (UDG) treatment
ROX™ passive reference dye
Two-step qRT-PCR
RNA template
RT efficiency
RT primers
Primers for the qPCR step
Minus RT control
Standards
Reference genes
qPCR
Template preparation and quality
Purity of nucleic acid templates is particularly important for qPCR, as contaminants may interfere with fluorescence detection. Most commercial DNA purification kits give satisfactory results for qPCR. Prior to amplification, the concentration of DNA and RNA should be determined by measuring the absorption at 260 nm (A260) in a spectrophotometer. The template amount needed for DyNAmo™ qPCR Kits depends on the type and quality of the template. In general do not use more than 500 ng genomic DNA in a 50 µl reaction.
Quantitation
Absolute quantitation is performed by plotting samples of unknown concentration to a standard curve generated from a dilution series of template DNA of known concentration. Typically the standard curve is a plot of the threshold cycle, C(t) against the logarithm of the amount of DNA. A linear regression analysis of the standard plot is used to calculate the amount of DNA in unknown samples. The slope of the equation is related to the efficiency of the PCR reaction. The PCR efficiency should be the same for the standards and the samples for quantitation to be accurate. PCR efficiency of the samples can be determined by doing a dilution series of these samples.
For a graph where C(t) is on the y-axis and log(DNA copy#) on the x-axis:
The slope of -3.322 equals 100% efficiency.
For a graph where log(DNA copy#) is on the y-axis and C(t) on the x-axis:
The slope of -0.301 equals 100% efficiency.
Relative quantitation is used to determine the ratio between the quantity of a target molecule in a sample and in the calibrator (calibrator being e.g. healthy tissue or untreated cells). The most common application of this method is the analysis of gene expression, e.g. comparisons of gene expression levels in different samples. Target molecule quantity is usually normalized with a reference gene.
If the amplification efficiency of a reference gene is the same as the efficiency of the target gene, comparative ∆∆C(t) method can be used for relative quantitation. Both the sample and the calibrator data isfirst normalized against variation in sample quality and quantity. Normalized values, ∆C(t)s, are first calculated from following equations:
∆C(t)sample = C(t)target - C(t)reference
∆C(t)calibrator = C(t)target - C(t)reference
The ∆∆C(t) is then determined using the following formula:
∆∆C(t) = ∆C(t)sample –∆C(t)calibrator
Expression of the target gene normalized to the reference gene and relative to the calibrator
= 2-∆∆C(t).
PCR primers
Careful primer design is important to minimize non-specific primer annealing and primer-dimer formation. The SYBR® Greenfifluorescence increases upon binding of the dye to any double-stranded DNA. Primer-dimer formation and non-specific binding can affect amplification efficiency with all chemistries. Standard precautions must be used during primer design to avoid primer-dimer or hairpin loop formation. Most primer design software tools will yield well-designed primers for use in qPCR. In most cases, good results are obtained using a concentration of 0.5 µM for each primer. The optimum primer concentration is usually between 0.1 and 1 µM.
MgCl2 optimization
Generally, it is not necessary to optimize the MgCl2 concentration with the DyNAmo qPCR Kits. Excessive MgCl2 concentrations can lead to amplification of nonspecific products and primer-dimers. Usually no more than 5 mM MgCl2 is required by any amplicon.
UNG (UDG) treatment
Due to the high sensitivity of qPCR even minute amounts of contaminating DNA can lead to false positive results. If dUTP is used in all qPCR reactions, the carry-over contamination from previous PCR runs can be prevented by treating the reaction samples with UNG prior to PCR. All Finnzymes’ DyNAmo Kits contain dUTP. UNG (uracil-N-glycosylase) digests dU-containing DNA, and the digested DNA cannot act as a template in qPCR (Longo M.C. et al. (1990) Gene 93: 125-128). UNG is inactivated during the first denaturation step in PCR. The UNG treatment step (50°C for 2 min) has no negative effect on qPCR performance when using hot start DNA polymerase, which is not reactivated at 50°C. To minimize contamination risk in general, tubes or plates containing reaction products should not be opened or analyzed by gel electrophoresis in the same laboratory area which is used to set up reactions.
ROX™ passive reference dye
ROX™ passive reference dye is used to normalize for non-PCR related fluorescence signal variation. Passive reference dye does not take part in the PCR reaction and its fluorescence remains constant during the PCR reaction. The amount of the ROX passive reference dye needed can vary depending on the type of the excitation. Note that the use of ROX passive reference dye may not be possible in combination with some fluorescent dyes.
Quick guides on how to use DyNAmo kits with ABI instruments that require ROX passive reference dye:
DyNAmo™ Flash SYBR® Green qPCR Kit (F-415)
DyNAmo™ HS SYBR® Green qPCR Kit (F-410)
DyNAmo™ Flash Probe qPCR Kit (F-455)
DyNAmo™ Probe qPCR Kit (F-450)
Two-step qRT-PCR
RNA template
Total RNA, mRNA, viral RNA or in vitro transcribed RNA can be used as a template for reverse transcription. The integrity and purity of the template RNA is critical for a successful cDNA synthesis. The RNA preparation should be free of any DNA or RNase contamination. The purity of RNA can be determined by measuring the ratio of A260/A280. The optimal ratio is 1.8-2.0.
RNA isolation should be performed under RNase-free conditions. Furthermore, any contamination with RNases from other potential sources like glassware, plasticware and reagent solutions has to be avoided by wearing gloves and using sterile tubes and pipet tips. Water used for the reactions should also be RNase-free, but not DEPC-treated, because traces of DEPC can inhibit PCR.
DNA contamination can be removed from the RNA sample by treating the sample with RNase-free DNase I. This should be done especially if primers for the qPCR step cannot be designed in exon-exon boundaries or in separate exons.
RT efficiency
The cDNA synthesis step is very critical in qRT-PCR. The efficiency of reverse transcription varies and can be low in some cases. The expression level of the target RNA molecule and the efficiency of the RT reaction must therefore be considered when determining the appropriate amount of starting template for subsequent PCR steps. The volume of cDNA template should not exceed 10 % of the qPCR reaction volume, as elevated volumes of template may reduce the efficiency of the PCR amplification. A dilution series of the template can be made to optimize the amount of the starting material used.
RT primers
Either specific primers, random hexamers or oligo(dT) primers can be chosen for the RT reaction. Specific primers decrease background, whereas random hexamers and oligo(dT) primers are useful if several different amplicons need to be analyzed from a small amount of starting material.
When choosing primers for cDNA synthesis, a good starting point is to use random hexamers. They transcribe all RNA (mRNA, rRNA, tRNA and in vitro transcribed RNA) producing cDNA that covers the whole transcript.
Oligo(dT) primers can be used to transcribe poly(A)+ RNAs. These include eukaryotic mRNAs and retroviruses with poly(A)+ tails. A number of different mRNAs are reverse transcribed, allowing subsequent qPCR detection of different targets from the same cDNA synthesis reaction. If the amplicon is located at the 5’ end of the transcript, random hexamers are recommended.
Gene-specific primers are used to transcribe only the particular RNA of interest, in contrast to oligo(dT)/random primers that transcribe all mRNAs/RNAs in the sample.
Primers for the qPCR step
PCR primers in qRT-PCR experiments should be designed to either anneal to sequences in two exons on opposite sides of an large intron or primers can be designed to anneal to the exon-exon boundary of the mRNA. This design enables differentiation between amplification of cDNA and contaminating genomic DNA, because with such primers, amplification of genomic DNA will be highly inefficient.
Minus RT control
A minus RT control should be included in all qRT-PCR experiments to test genomic DNA contamination. It contains all the reaction components except the reverse transcriptase. This control reaction should be free from reverse transcribed cDNA. If PCR amplification is detected, it is most likely derived from contaminating genomic DNA.
Standards
Using RNA molecules as standards for RNA quantification is recommended. The use of RNA standards takes the variable efficiency of the reverse transcription into account. RNA standards can be generated for example by cloning the cDNA of interest to a vector containing RNA polymerase promoter, e.g. T7 or Sp6. From the vector, the insert can be in vitro transcribed to obtain thefinal RNA standard with identical sequence than the target amplicon. The vector must then be degraded with RNase-free DNase, and the concentration of the RNA standard determined spectrophotometrically. Alternatively, a defined RNA preparation, e.g. from a cell line or a virus, with known concentration can be used as an RNA standard.
Reference genes
When studying gene expression, the quantity of the target gene transcript needs to be normalized against the quantity of a reference gene transcript in the same sample. Examples of commonly used reference genes are β-actin, GAPDH and 18S rRNA. A gene used as a reference should have a constant expression level independent of the variation in the state of the sample tissue. A problem is that even housekeeping genes are to some extent variable in their expression. That is why several reference genes are usually required, and their expression needs to be assayed in each experiment.
The amplification efficiency of a reference gene should be the same as the efficiency of the target gene. If this is not the case, the results have to be corrected for the efficiency.
Since RNA quantification involves a number of variables, and each experiment is inherently different, careful experimental design is very important. Useful information and guidelines for experimental design, normalization, RNA standards, etc. can be found in the following review articles:
Bustin S.A. (2000) Journal of Molecular Endocrinology 25: 169-193.
Bustin S.A. (2002) Journal of Molecular Endocrinology 29: 23-39.
