在RT-PCR中避免DNA污染（Avoiding DNA Contamination in R

A frequent cause of concern among investigators performing quantitative RT-PCR is inaccurate data due to DNA contamination in RNA preparations. Although DNA contamination is easily detected by performing a "no-RT" control, there is no easy remedy.

In this technical bulletin, we present data showing levels of DNA contamination in RNA generated by different procedures, and suggest several precautionary measures that can be implemented to reduce the impact of this persistent problem.

RT-PCR and Genomic Contamination

RT-PCR is an increasingly popular method for the quantitative analysis of gene expression. With this popularity comes a heightened awareness that most techniques used for total RNA isolation yield RNA with significant amounts of genomic DNA contamination.

PCR cannot discriminate between cDNA targets synthesized by reverse transcription and genomic DNA contamination. At Ambion, we can routinely perform PCR from residual genomic DNA present in total RNA samples isolated by most commonly used techniques.

To illustrate this problem, we performed RT-PCR on mouse liver RNA isolated by a multi-step guanidinium thiocyanate/acid phenol:chloroform extraction (ToTALLY RNA™), a one-step extraction (TRI Reagent®), a filter-binding based extraction (RNAqueous®), by centrifugation through a CsCl cushion, and by two rounds of oligo d(T) selection using Ambion's Poly(A)Pure™ Kit (see Figure 1a).

Regardless of whether reverse transcriptase was added in the reverse transcription step, gene specific product is synthesized in most samples. Among the total RNA samples, the amount of DNA contamination is lowest in the CsCl-pelleted RNA.

No signal is apparent in the oligo d(T)-selected sample. The PCR products in the "no-RT" samples are the result of amplification from trace amounts of genomic contamination.

Figure 1. DNA Contamination in RNA Isolated by Five Different Methods. Mouse liver total RNA was isolated according to protocol by five different methods. 0.5 µg RNA was used in RT-PCR reactions with Ambion's RETROscript® Kit.

PCR reactions were performed with 5 µg RNA. 10 µl of each reaction was electrophoresed on a 2% agarose gel and stained with EtBr.

Differential Enrichment by Oligo d(T) Selection

Although two rounds of oligo d(T) selection are sufficient to remove genomic DNA contamination, there are two drawbacks to using this technique to control for DNA contamination. First, oligo d(T) chromatography is expensive and labor intensive for routine analysis.

Secondly, a potentially serious problem not usually addressed is that relative amounts of individual transcripts can change with oligo d(T) chromatography, probably as a result of differential polyadenylation between tissues or in response to stimuli.

At Ambion, we have found that oligo d(T) selection can even change the apparent abundance of transcripts from genes that are thought to have invariant expression.

For example, when we compare the relative enrichment of cyclophilin and GAPDH transcripts by Northern blot analysis of total versus oligo d(T) selected mouse RNA, we see an obvious change in the apparent abundance of these two transcripts.

As shown in Figure 2, oligo d(T) selection enriches GAPDH and cyclophilin 17X and 22X, respectively, from kidney RNA, but 21X and 28X from thymus RNA. The source of this variation is unclear, but the implications for quantitation from oligo d(T) selected RNA are impossible to ignore.

Figure 2. Differential Enrichment of Specific mRNAs by Oligo dT Chromatography. A Northern blot containing total RNA (1 µg) and twice oligo d(T) selected RNA (50 ng) from mouse thymus and kidney was hybridized simultaneously with GAPDH and cyclophilin RNA probes. Hybridization signals were quantitated with a Bio-Rad Molecular Imager.

Primer Design

Primers for quantitative experiments are typically designed to amplify a target between 150 and 600 base pairs. Targets smaller than 200 bp are difficult to resolve on agarose gels, and larger targets place a greater burden on the investigator to optimize PCR conditions. The critical aspect for RT-PCR primer choice with respect to minimizing the problems associated with DNA contamination is to design primers that span at least one intron of the genomic sequence. This will result in a PCR product from genomic contamination that will be larger in size than the product generated from the cDNA. In fact, primers can be designed to span a sufficiently large genomic fragment such that amplification from contaminating DNA may be not be possible. For genes in which the genomic sequence is published, the positions of the splice junctions can be found by retrieving the sequence from the (责任编辑：大汉昆仑王)