Abstract
The presence of contaminating genomic DNA (gDNA) in RNA preparations is a frequent cause of false positives in RT-PCR-based assays aimed at gene expression analysis. Sometimes this phenomenon cannot be avoided even when specific precautions in the assay design are taken (e.g. design of intron-spanning primers), for example, when the target mRNA presents pseudogenes at the DNA level. For these reasons, the inclusion of a DNase digestion step is often necessary. In this chapter two alternative methods for RNA treatment with DNase are described. The choice of the most suitable method is largely dependent on the availability of starting RNA. The conventional DNase treatment requires a step of phenol/chloroform. This method is useful when an RNA solution is not pure and large RNA amounts are available, since this treatment causes about 50% RNA loss. The DNase digestion followed by enzyme heat inactivation is particularly suitable when an RNA starting quantity is very low because, theoretically, no further RNA is lost during heat treatment. This method may be very useful when an RNA has been extracted from small biopsies or cytologic specimens.
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Notes
- 1.
Pseudogenes are DNA non-functional sequences present in the genome that have strong similarities to mRNA but, in general, are unable to be transcribed. The nonfunctionality of the pseudogenes is often caused by the lack of functional promoters or other regulatory elements. Pseudogenes are quite difficult to identify, because their characterization is performed through sequence calculations and alignments rather than biologically proven. A comprehensive list of identified pseudogenes can be found at http://www.pseudogene.org.
- 2.
Sterile, disposable plasticware should preferably be used because it is RNase free. If general laboratory glassware or plasticware is used, it should be pre-soaked in 0.1% DEPC-treated H2O for 2 h at 37°C. The DEPC-treated items should be rinsed thoroughly with DEPC H2O and then autoclaved. Alternatively, glassware could be put in an oven at 250°C for 2 h.
- 3.
After this treatment the water is essentially free from RNases and can be used to prepare solutions or to rinse any items to use for RNA isolation. DEPC is a carcinogen and should be handled with care under a fume hood.
- 4.
Clean the pipettes with a disinfectant (e.g., Meliseptol®rapid) and leave them under the UV lamp for at least 10 min. Alternatively it is possible to autoclave the pipette depending on the provider instructions. Pipettes dedicated only to RNA extraction and treatment should be preferably used.
- 5.
If more than 5 μg RNA is digested, increase the DNase units. If a larger amount of RNA is treated, scale up the reaction volume because the viscosity of the solution may increase, preventing DNase activity.
- 6.
The volume of reaction can be increased up to 100 μl when, for example, the starting RNA is resuspended in large volumes; in such case, the DNase unit concentration has to be maintained by increasing the quantity.
- 7.
Alternatively, it is possible to purify DNase-treated RNA by the use of silica columns or the direct performance of an on-column DNase digestion (for details, see RNase free DNase set on http://www.qiagen.com/).
- 8.
See Chap. 16 for details.
- 9.
Sterile, disposable plasticware should be preferably used because it is RNase free. If general laboratory glassware or plasticware is used, it should be pre-soaked in 0.1% DEPC-treated H2O for 2 h at 37°C. The DEPC-treated items should be rinsed thoroughly with DEPC H2O and then autoclaved. Alternatively, glassware could be put in an oven at 250°C for at least 2 h.
- 10.
After this treatment, the water is essentially free from RNases and can be used to prepare solutions or to rinse any items to be used for RNA isolation. DEPC is a carcinogen and should be handled with care under a fume hood.
- 11.
The EDTA helps to protect RNA at high temperatures.
- 12.
Clean the pipettes with a disinfectant (e.g., Meliseptol®rapid) and leave them under the UV lamp for at least 10 min. Alternatively it is possible to autoclave the pipette according to the provider instructions. Pipettes dedicated only for RNA extraction and treatment should be preferably used.
- 13.
If more than 4 μg RNA are digested, increase DNase units. If a larger amount of RNA is digested, scale up the reaction volume as more RNA may increase the viscosity of the solution preventing DNase activity.
- 14.
The volume of reaction can be increased up to 100 μl when the starting RNA is resuspended in large volumes; in this case also the DNase units must be increased.
- 15.
This incubation time and temperature has been shown to be sufficient to remove contaminant DNA (manufacturer’s protocol suggests 10–20’ at 37°C).
- 16.
For spectrophotometric quantification of RNA after DNase treatment remember to use a DNase solution complete of buffer and EDTA without RNA as a blank.
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Dotti, I., Bonin, S. (2011). DNase Treatment of RNA. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_18
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DOI: https://doi.org/10.1007/978-3-642-17890-0_18
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