Avoiding DNA Contamination in RT

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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.

Lane

RNA Isolation Method

1

One Step RNA Isolation (Tri Reagent)

2

Glass Binding Method (Ambion"s RNAqueous™ Kit)

3

Acid Phenol Chloroform Method (Ambion"s ToTALLY RNA™ Kit)

4

CsCl cushion

5

Oligo dT Selection (Ambion"s Poly(A)Pure™ Kit)

6

H2O Control

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.