INFORMATION - This page and the following pages provide in-depth information on RT-PCR

 

RT-PCR

RT-PCR (reverse transcription-polymerase chain reaction) is a sensitive and powerful tool for the detection and quantification of mRNA expression. In fact, this technique is sensitive enough to enable quantification of RNA from a single cell. Traditionally, RT-PCR involves two steps: the RT reaction and PCR amplification. RNA is first reverse transcribed into complementary DNA (cDNA) using an enzyme, reverse transcriptase. The resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes. RT-PCR can also be carried out as one-step RT-PCR in which all reaction components are mixed in one tube prior to initiation of the reaction. Although one-step RT-PCR offers simplicity and convenience and minimizes the possibility for contamination, the resulting cDNA cannot be used for detecting multiple messages from a single RNA sample as in two-step RT-PCR.

In comparison to traditional approaches (i.e., Northern blot, dot blot and hybridization protection assays and in situ hybridization) RT-PCR-based assays are the most common method for characterizing or confirming gene expression patterns and comparing mRNA levels in different sample populations [1].

 

Since the first reports of quantitative RT-PCR [2], the use of this technique has grown at an exponential rate. Despite the power and relative ease growth, the ability of RT-PCR to accurately quantify mRNA levels is hampered by a technical hurdle. Specifically, as the PCR greatly amplifies the target, errors are also amplified. As a result, variability can be very large and preclude accuracy and reliable quantification. For this reason, quantitative RT-PCR is viewed with skepticism by many investigators and reviewers. Such concern can be obviated by thoughtful experimental design and carefully validating the technique for a given gene or a given laboratory.

 

The most promising innovation applied to conventional RT-PCR protocols is the development of standardized, quantitative, competitive RT-PCR [3], which is also known as Standardized RT-PCR (StaRT). StaRT addresses some of the major problems associated with conventional competitive RT-PCR protocols: the use of standardized competitor templates allows comparison between experiments and the use of
internal standards solves problems of variation in template starting amounts and operator loading errors [4] The method uses standardized primer and internal competitor template sets that are tailored individually for each target gene. These amplify with the same kinetics as the target under investigation, and generate conventional RT-PCR products. Target gene mRNA levels are measured relative to their respective competitor templates and are normalized to reference genes to control for cDNA loaded into the reaction. This result in the reporting of each gene expression measurement as a numerical value that allows direct comparison between experiments carried out within the same or different laboratories.

 

The design of the assay allows the analysis to be performed at the endpoint of the RT-PCR reaction and it can be automated through the use of capillary electrophoresis or micro-channel electrophoresis instruments. At the moment, there are hundreds of such target/competitor sets available and all can be multiplexed in the same reaction. The intriguing aspect of this standardization is that a common databank has been set up that will eventually allow the consolidation of individual results into what have been termed interactive gene expression indices [5]. This system, together with the databank, has been commercialized. The main disadvantage of the system is probably the inflexibility inherent in a system that is limited to sets of primers available from one supplier. Furthermore, it does not eliminate the errors associated with individuals carrying out the reactions.

 

 

RT-PCR steps
The advent of PCR in 1986 [6] and the combination of reverse transcription and PCR [7] quickly led to the use of RT-PCR for mRNA quantification [8]. Ever since its advent, quantitative RTPCR technology is still being optimized. Figure 1 presents the steps in a quantitative RTPCR approach.

 

Figure 1. Representative flowchart of qRT-PCR

 

 

 

Step I - RT
Isolation of RNA (either total RNA or polyadenylated RNA) from samples can be accomplished using a number of methods [most commonly with a guanadinium-based chaotropic agent [9]]. The initial step in RT-PCR is the production of a single-strand cDNA copy of the RNA through the action of the retroviral enzyme, reverse transcriptase. Two main types of enzyme are commercially available: Moloney murine leukemia virus (MMLV-RT) and avian myeoblastosis virus (AMV-RT). The choice is largely a matter of personal preference or cost, but some direct comparisons have been reported [10].

 

An oligonucleotide primer is required to initiate cDNA synthesis. The primer anneals to the RNA, and the cDNA is extended toward the 5¢ end of the mRNA through the RNA-dependent DNA polymerase activity of reverse transcriptase. Primers can be either gene-specific or nonspecific; both have advantages and disadvantages. Random hexamer primers contain all possible nucleotide combinations of a 6-base oligonucleotide and bind to all RNAs present. Similarly, oligonucleotides consisting solely of deoxythymidine residues [oligo(dT)] anneal to the polyadenylated 3¢ tail found on most mRNAs. RT reactions primed by random hexamers and oligo(dT) primers can be split into a number of different PCRs, each with different gene-specific primers. This method maximizes the number of genes that can be assayed from a small RNA sample.

 

Alternatively, a gene-specific primer can be used for the RT reaction. For some genes, especially rare messages, the use of sequence-specific primers increases specificity and decreases background associated with other types of primers. These gene-specific RT primers work well in conjunction with elevated RT reaction temperatures to eliminate spurious transcripts [11]. These (antisense) primers can then be used for the subsequent PCR in conjunction with the corresponding gene-specific forward (sense) primer [12].

 

The RT step is the source of most of the variability in a quantitative RT-PCR experiment. The reverse transcriptase enzyme is sensitive to salts, alcohols or phenol remaining from the RNA isolation. Compounding this problem, a biphasic relationship between salt and RT-PCR amplification has been observed. Under some conditions, low concentrations of added salt (< mM) enhance signal, and high levels of salt (³50 mM) decrease the output signal. Therefore, salt contamination, carried over from the RNA precipitation step, can affect the apparent RNA levels. Inter-tube and inter-experiment variability are therefore common for RT reactions. This signal output variability is a central issue in quantitative RT-PCR. It cannot be assumed that different reactions have the same RT efficiency. If one can minimize the nonspecificity and variability in this step, then the reliability of the ensuing quantification will be optimized.

 

Step II - PCR
Following the RT reaction, the cDNA is amplified by PCR. The PCR is usually carried out using an aliquot of the RT reaction or by adding the necessary PCR components directly to the RT reaction [12]. PCR is generally a three-step process, with denaturation, annealing and elongation steps, with temperatures that vary and are subject to a number of considerations that should be determined empirically. The number of cycles depends on the amount of target present and the efficiency of the reaction. It is best to optimize cycle number to produce easily visualized products while remaining out of the plateau phase of the reaction.

 

Primer design and selection is also of primary importance in the PCR step. For instance, designing intron-spanning primers for PCR allows DNA contamination to be assessed. With this design, intron-containing DNA will give a different amplification product than will a spliced RNA. For intron-less genes, it is necessary to perform a control RTPCR in which the reverse transcriptase is omitted. In fact, this valuable "minus-RT" control should be used in all experiments. The common problem of amplification product contamination from previous PCRs can be detected in this way.

 

Read More 1. RT-PCR 2. PCR-Detection & Monitoring 3. Competitive & Non Competitive PCR 4. PCR Protocols 5. Quantification , 6. References