Variables to RT-PCR Success
One key variable to the success of any RT-PCR experiment is RNA isolation. RNA isolation methods such as acid phenol extractions, glass fiber filter purifications, and single-step reagents can each provide RNA of acceptable quality (though researchers must still be concerned with possible protein, phenol and alcohol carryover, all of which can compromise downstream enzymatic reactions). The one issue all RNA isolation methods have, however, is the inability to completely remove genomic DNA contamination from the RNA sample. DNA removal is critical especially for RT-PCR applications since DNA can serve as a template during the PCR portion of the experiment, resulting in false positives, background, etc.
All RNA Isolation Methods Yield RNA Containing Residual Genomic DNA
Ambion has found that no RNA isolation method consistently produces RNA free of residual genomic DNA. To illustrate this, RT-PCR was performed on mouse liver RNA isolated by several different methods (Figure 1). Results in Figure 1 show that, regardless of the isolation method employed, gene specific product is synthesized in the absence of reverse transcriptase, indicating that none of these RNA isolation methods produce RNA free of residual genomic DNA. Because RNA free of DNA contamination is important, Ambion has established rigorous quality control standards for the treatment and testing of the FirstChoice™ Total and Poly(A) RNAs to ensure that DNA contamination is minimized.
Figure 1. DNA Contamination in RNA Isolated by Five Different Methods.
Mouse liver total RNA was isolated according to protocol by five different methods and 0.5 µg RNA was used in RT-PCR reactions with Ambion's RETROscript™ Kit
. PCR reactions (minus-RT) were performed with 5 µg RNA. 10 µl of each reaction was electrophoresed on a 2% agarose gel and stained with EtBr. (Lane 1
) One Step RNA Isolation (Lane 2
) Glass Binding Method (Lane 3
) Acid Phenol Chloroform Method (Lane 4
) CsCI cushion (Lane 5
) Oligo dT Selection (Lane 6
Getting Rid of Contaminating DNA
There are a variety of methods to reduce or eliminate DNA contamination in RNA preparations and Ambion has tested several methods to determine which is most effective. These methods include DNase I digestion, acid phenol:chloroform extraction, and lithium chloride precipitation. The details and results of these tests can be found in Ambion Technical Bulletin 181, "Methods to Remove DNA Contamination from RNA Samples
DNase I Treatment is Most Effective
DNase I digestion has consistently proven to be the most effective method for removing DNA contamination from RNA samples. Figure 2 demonstrates how effective DNase I treatment can be, as analyzed by real-time RT-PCR analysis. Samples post DNase I treatment were analyzed for genomic DNA contamination using a TaqMan® GAPDH assay. Results indicate that samples containing as much as 15% DNA by mass were essentially free of DNA after DNase treatment.
Figure 2. Effect of DNase I Digestion on CT Values.
100 ng of total RNA isolated from 3 different human tissues were used directly in a 40 cycle PCR reaction using TaqMan probes for GAPDH.
Simplifying RNA Isolation for RT-PCR
Ambion has simplified RNA isolation for RT-PCR by combining a fast, phenol-free method of RNA isolation with an effective DNase I treatment, including a novel reagent to clean up the DNase reaction. The RNAqueous™-4PCR Kit utilizes Ambion's RNAqueous technology to purify total RNA on glass fiber filters in a microfuge tube format. The RNA is then eluted and digested with Ambion's RNase-free DNase I to rid the sample of genomic DNA contamination. Following DNase I digestion, a novel reagent, DNA-free™ DNase Removal Reagent, is added to the sample. This reagent removes the DNase I and divalent cations rapidly and effectively, eliminating the need to heat inactivate DNase I, which can lead to strand scission of the RNA. It also reduces protein contamination of the sample. Removing DNase I by phenol:chloroform extraction, which can compromise the yield from limiting RNA samples, becomes unnecessary. Figure 3 demonstrates the effective removal of genomic DNA from RNA samples isolated from five different tissue types using RNAqueous-4PCR.
Figure 3. RT-PCR Experiments Using Total RNA Isolated with the RNAqueous™-4PCR Kit.
RNA samples (5 µl) were used as templates for reverse transcription reactions and then 10 percent of the resulting cDNA was amplified by PCR using S15 primers. The lanes to the left of the markers are PCRs done without reverse transcription, demonstrating the lack of genomic DNA contamination in these RNA samples. The lanes to the right of the markers show the S15 RT-PCR product from the indicated samples