It should be possible to choose a primer combination that yields either no product (additional intron sequence produces too long a target for efficient PCR) or an easily distinguishable product when amplifying from genomic DNA. An additional wrinkle is that pseudogenes exist in the mammalian genome for many genes, including the most commonly used internal controls (ß-actin, GAPDH, cyclophilin). These sequences, arising from integration of a reverse transcription product into the genome, do not have introns. Thus, the size of a PCR product amplified from a pseudogene may be identical to that produced from a cDNA copy. The only way to identify these products is to perform a "no-RT" control as shown in Figure 3. The true product from RNA is 361 base pairs. The "no-RT" control yields both a fragment identical in size to the expected RT-PCR product from the RNA transcript (from a pseudogene), and a 1.2 kb fragment from the legitimate genomic locus. If it is absolutely essential to avoid amplification from these sequences, an amplified fragment from a pseudogene may be sequenced, and primers designed to regions where the sequence varies from the legitimate copy of the gene. As little as a one or two nucleotide difference at the 3' end of a primer binding site can completely inhibit amplification from the pseudogene.
| Figure 3. DNA Contamination in RNA. Mouse liver total RNA was isolated according to protocol. RT-PCR reactions were performed using Ambion's RETROscript® Kit and 0.5 µg RNA. PCR reactions were performed with 5 µg RNA. 10 µl of each reaction was electrophoresed on a 2% agarose gel and stained with EtBr. |
DNase I Treatment
In a recent informal survey of RT-PCR users, we found that the field is evenly divided by those users who believe that DNase I treatment solves the problem of genomic DNA contamination and those who feel that DNase I treatment is an unacceptable solution. Detractors claim that DNase I treatment and the subsequent inactivation steps compromise the performance of their RT-PCR reactions to an unacceptable degree. Much of the problem these users experience may be traced to the extreme temperatures used to inactivate the DNase I prior to reverse transcription. Huang, et al. (Biotechniques, (1996) 20:(6)1012-1020) report complete inactivation of DNase I by heat denaturation at 75°C for 5 minutes. Lower inactivation temperatures do not completely inactivate DNase I, while higher temperatures appear to damage the RNA template. DNase I treatment followed by heat inactivation is a simple enough technique for routine use in systems in which genomic DNA contamination is a problem. The use of high quality, RNase-free DNase is crucial. Two additional conventional methods of reducing contaminating genomic DNA from total RNA preparations are acid phenol extraction, which partitions DNA into the organic phase, and LiCl precipitation, which selectively precipitates RNA from solution (protein and DNA remain in the supernatant). A description of these techniques can be found in Ambion's Technical Bulletins #158 and #160. These techniques can be used after DNase I treatment to inactivate the enzyme and precipitate the RNA prior to reverse transcription. Finally, it should be noted that DNase I treatment neither relieves the investigator of the burden of sensible primer design, nor of the necessity to perform the appropriate "no-RT" controls.
