1. Common priming strategies for RT reactions include using gene-specific primers, random hexamers, or oligo-dT 12-18 mers. I have had best results with oligo-dT, and this has the further advantage of allow ing the RT product to be assayed for several transcripts, either simultaneously or sequentially.
The RNA yield from 10 mg tissue should be at least 1 pg. This should allow a 30-pL "large-scale" RT reaction; if less tissue is used or if the gene of interest is expressed at very low levels, a 7.5-pL "small-scale" reaction can be used. If PCR is to be performed using primers from within one exon, duplicate reactions should be set up with and without reverse transcriptase.
2. Ideally, primers to be used for PCR will correspond to two different exons, so that genomic DNA will give a larger product than cDNAs from the transcript being assayed (or no product at all where large in-trons are involved). Where both primers are of necessity within one exon, the RNA preparation must be treated with DNase I, so that PCR bands will indicate mRNA expression and not contaminating DNA.
3. The products of RT reactions can be used directly in PCR reactions with no further purification. Usually one-sixth of a large-scale RT reaction is amplified or the whole of a small-scale RT reaction; the amounts can be adjusted according to requirements.
4. 7"is an annealing temperature suitable for the primers used. It should be determined empirically, but is usually between Tm - 10 and Tm + 10 (°C), where:
5. These extension times at 72°C are suitable for products up to 1 kb. They should be increased 30 s for each additional kilobase.
6. A suitable 5X loading dye for agarose gels that does not obscure PCR products is. 10 mMTris-HCl, pH 7-8.5, 1 mMEDTA, 20% Ficoll, and Orange G to taste.
7. Improving sensitivity: A number of simple strategies can be used to manipulate the sensitivity of detection. These include:
a. The number of cycles can be increased. Some workers have used up to 60 cycles, but depending on conditions and reagents used, it is doubtful that much enzyme activity would survive this punishment. Also, the amount of specific product can plateau beyond a certain number of cycles, favoring the amplification of undesired products.
b. Enzyme can be supplemented after a suitable number of cycles (say 20). The amount of work to be done by the polymerase doubles with each cycle, yet enzyme activity decreases with each denaturation step c. The agarose gel can be blotted onto a nylon filter that can then be hybridized to a radioactive probe for the gene of interest. The probe can include both primer sequences, since the short length of probe/ primer homology will not permit probe binding to undesired "ghost" PCR bands at high stringency, d. A second round of PCR can be undertaken using a second pair of primers nested within the first pair. For example, after 20 cycles in the first round, 1 pL is transferred into a second reaction containing the nested primers, and 20 further cycles executed.
The paradox of all these strategies is that the greater the sensitivity, the greater the risk of revealing a false positive caused by contamination, thus invalidating the experiment. Great care needs to be taken to avoid this problem (see Note 10).
8. Quantitation: Applied to studies of gene expression, quantitative PCR is an attempt to measure the abundance of transcripts in a tissue using the amount of final PCR product as a guide. A number of properties of PCR mean that quantitation is full of pitfalls. For example, because PCR proceeds exponentially, a small difference in amplification efficiency between two otherwise identical samples will yield vastly different amounts of product Many regard PCR as semiquantitative at best.
One factor required for quantitation is an exact exponential relationship between the number of cycles and the amount of product. It is generally held that this relationship breaks down beyond 15-20 cycles, depending on conditions. Beyond this, the amount of abundant product begins to plateau, whereas less abundant products may continue to be amplified exponentially.
Another problem is tube-to-tube variability. This can only be overcome by multiple independent assays of the same starting tissue. Clearly, this will magnify the work load of the experiment severalfold, but without this extra effort, attempts at quantitation are meaningless.
The most effective way to measure product is to use a radioactive label. Radioactivity can be measured after removal of unincorporated label from the product or after excision of the product from a gel. This should always be related back to a standard curve made using different dilutions of a control RNA sample.
9. Using PCR to distinguish closely related genes. To distinguish between the gene of interest and any closely related genes (for example, a heterologous transgene vs its endogenous counterpart), at least one nucleotide must differ between the two transcripts. Normally, there will be many more differences, for example, in the 5'- or 3'-untranslated regions of the transcripts, that will allow the design of primers that will specifically recognize only the cDNA of interest
Where the number of nucleotide differences is limited, these should be incorporated into the 3' end of the oligonucleotides, to reduce the possibility of false priming. The specificity enhancer Perfect Match (Stratagene, La Jolla, Ca) may be useful in these situations, but this reagent must be carefully titrated so as not to inhibit amplification.
Often, differences between two genes will create or abolish a restriction enzyme recognition site. If this is the case, the primers can be designed to amplify both gene products, which can then be distinguished on an agarose gel after the appropriate restriction digest. If all else fails, sequencing of PCR products can be used to identify which of two closely related genes is expressed.
10. Safeguards and controls: The exquisite sensitivity of PCR is at once its greatest asset and its greatest problem. Unless painstaking care is used, contamination is almost certain to arise, causing false positives and ruining experiments.
The specific DNA molecule being amplified poses the greatest threat. Such vast quantities of these molecules are being produced that it is easy for some of them to find their way into PCR reagents. Other dangers include plasmid subclones of the gene of interest, which are likely to be wafting around the lab and lurking on key pieces of equipment.
Safeguards against contamination are described in a recent Nature article (7). The importance of extreme care from the outset cannot be overemphasized. The highest priority is physical separation of the PCR setup area from the normal lab area where PCRs are run and analyzed. Preferably dedicated equipment—in particular, pipets, centrifuges, and freezers—should be used. It is helpful to UV-irradiate pipets between experiments. Reagents should be made up from stock powders and solutions especially reserved for PCR. Buffers and primers should be aliquoted when made up, and aliquots changed frequently. Gloves should be changed often, particularly after touching anything other than your PCR equipment.
Each assay should, of course, include a positive control that may be a known expressing tissue or some purified RNA from such tissue. It is also essential to include a nonexpressing (e.g., nontransgenic) tissue or perform a mock analysis of water, as a negative control.
It is useful to include primers for an irrelevant, but ubiquitously expressed gene, in addition to the primers for the gene of interest, as a control for the quality and quantity of RNA in each sample. I use the following primers for mouse hypoxanthine phosphoribosyltransferase (Hprt):
Hprt. 1 a: 5'-CCTGC TGGAT TACAT TAAAG CACTG-3' Hprt.lb: 5'-GTCAA GGGCA TATCC AACAA CAAAC-3' These primers have been used at annealing temperatures from 45 to 65°C, and so are compatible with almost any other primer set (8-10). They give a product of 354 bp, and span exons 3-8 of the Hprt cDNA (11). No PCR product is obtained from genomic DNA To ensure that they do not obscure the signal from the gene of interest, the Hprt primers can be used down to 25 ng/reaction.
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