DNA-free RNA Isolation
In commonly used RNA isolation methods, the absence of genomic DNA is still a challenge. Particularly for downstream quantitative RT-PCR (qRT-PCR, co-amplified genomic DNA can lead to nonspecific results.
1.) Detection of genomic DNA in RNA samples
Different techniques have been used to monitor contamination of RNA by genomic DNA. Large amounts of contaminating DNA can be detected using OD measurement (producing a 260/280 nm ratio far below the optimum value of 2), or via agarose gel electrophoresis, where very high molecular weight nucleic acid bandings are obtained.
Nevertheless, in sensitive downstream applications like qRT-PCR, genomic DNA traces, not detectable by the above methods, can also produce nonspecific results. This is because both reverse transcribed mRNA (cDNA), as well as the contaminating genomic DNA, can serve as template for the subsequent PCR amplification. The result of this co-amplification is one or more additional PCR fragments that can be visualized via agarose gel electrophoresis based on their distinct molecular weight (see Figure 1).
One strategy to avoid this co-amplification is a PCR primer design that span exon boundaries.
This primer design strategy includes one or more intronic sequence motifs in the genomic DNA shifting the amplification efficiency of the PCR reaction toward the cDNA template. The specificity of the qRT-PCR assay should be monitored using a reverse transcriptase minus control reaction in the qRT-PCR setup. This control reaction is treated and handled similar to the other qRT-PCR reactions, with the exception that instead of the reverse trancriptase enzyme, water is added to the reaction mixture. PCR fragments amplified in this control reaction indicate contaminating genomic DNA.
Figure 1: Overview of first strand cDNA synthesis with different types of RT primers.
A GAPDH (lanes 1, 3 and 5) and RP49 (lanes 2, 4 and 6) gene specific PCR was performed including a control reaction lacking the reverse transcriptase enzyme (lanes 5 and 6). The PCR fragments in lanes 5 and 6 indicated by arrows, are of higher molecular weight based on the included intron sequence, and therefore indicative of contamination of the RNA template with genomic DNA.
2.) DNase I treatment
For removal of genomic DNA from RNA samples, a DNase I treatment is recommended. The DNAse I enzyme is classified as an endonuclease which is able to digest single and double-stranded DNA into single bases or oligonucleotides. The enzyme is DNA specific without negative effect on the integrity of the remaining RNA (Vanecko and Lasbowski, 1961).
DNase I treatment can be performed at the same time as the RNA isolation. This convenient procedure is used in the High Pure RNA Isolation Kit. The DNase I enzyme is included in the kit, and is directly applied during the spin column isolation procedure. The RNA is bound to the silica fleece in the presence of chaotropic salts, and the DNase I is directly applied and incubated with the purified RNA on the spin column. The digested DNA is subsequently washed from the spin column, leading to pure RNA eluted from the spin column.
In order to protect the isolated RNA, the DNase I Enzyme used should be of RNase-free quality. The recombinant DNase I Enzyme, RNase-Free from Roche is not only guaranteed to be RNase-free according to the strict current quality control, but is furthermore free of any animal components, such as contaminating DNA.
In cases of very high levels of endogenous genomic DNA, treatment with DNase I during the isolation process can still leave residual contaminating DNA in the isolated RNA. In this case DNase treatment can be performed after the RNA isolation.
Tip: As a rule of thumb for the DNase I digestion, use one unit of DNase I per 1 to 5 μg of total RNA in a 50 μl total volume incubated for 20 minutes at +25 to +37°C.
After the additional DNase digestion step an additional purification of the RNA from the DNase I enzyme is mandatory. This purification can be done by a cleanup procedure using the High Pure RNA Isolation Kit following the kit protocol (2.2 Isolation of Total RNA from Cultured Cells). Prior of the cleanup procedure the sample volume should be increased to 200 μl using the elution buffer included in the kit.
Note that in this case, the on-spin column DNase digestion step should be omitted from the protocol mentioned in the package insert.
The main benefit of this method is its ease of use, lack of toxic substances as well as the high recovery of total RNA.
Figure 2: Genomic DNA digestion by DNase I treatment.
A GAPDH (lanes 1, 3, and 5) and RP49 (lanes 2, 4, and 6) gene-specific PCR was performed including a control reaction lacking the reverse transcriptase enzyme (lanes 5 and 6). The PCR fragments in lanes 5 and 6 indicated by arrows, are of higher molecular weight based on the included intron sequence, and therefore indicative of contamination of the RNA template with genomic DNA.
A) Amplification of a GAPDH fragment by PCR.
B) Minus-RT control reaction with and without DNase I treatment. RNA was isolated from 1 x 106 K562 cells (human lymphocytes) using the High Pure RNA Isolation Kit with a subsequent amplification of a GAPDH-specific PCR fragments (lanes 1 to 6). No unexpected PCR fragment were obtained, indicating the absence of genomic DNA in lanes 1 to 4 (+DNase treatment), whereas those samples lacking the DNase treatment showed PCR products produced from contaminating genomic DNA (lanes 5 and 6) serving as template in the PCR amplification.
Tip: Even after DNase treatment, a subsequent PCR should be performed using exon-overlapping primer design. Also a reverse trancriptase minus reaction should be included as a control reaction for monitoring amplification specificity.
3.) Incomplete DNase digestion.
Very high levels of endogenous genomic DNA can still leave residual contaminating DNA in the isolated RNA. In case an additional DNase digestion step with subsequent cleanup is not an option, the amount of starting material should be reduced (i.e., 106 cultured cells, 500 μl whole blood, 108 yeast cells or 109 bacterial cells are maximum for the High Pure RNA Isolation Kit).
Another possible cause for incomplete DNA digestion is that the activity of the DNase I Enzyme can be decreased by suboptimal buffer conditions. The DNase I enzyme requires different divalent cations, both Mg2+ as well as Ca2+, for optimal performance.
Tip: For optimal performance of the DNase I digestion the concentration of nucleic acid should be approximately 100 μg/ml.
Occasionally, the additional DNase I treatment, and specifically the heat inactivation of the DNase I enzyme, can negatively effect subsequent RT-PCR results. High temperatures necessary for the inactivation of the DNase I enzyme can also lead to RNA degradation. The heat inactivation of the DNase I enzyme requires an incubation at +75°C for 15 minutes. Since RNA molecules are heat sensitive (Huang et al., 1996), this incubation time should be as short as possible.
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