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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1 Introduction and Purpose
A frequent cause of false positives in RT-PCR-based assays is the amplification of genomic DNA (gDNA) contaminating RNA preparations. Because of PCR sensitiveness, a single copy of a genomic sequence can be theoretically detected. This phenomenon is exacerbated when tested genes present pseudogenes at the DNA level, which have sequences similar to mRNA (information about pseudogenes can be obtained in the webFootnote 1). For this reason DNA removal by DNase digestion is often a necessary step. Moreover, what has been observed is a decrement in real-time threshold cycles in cDNA obtained from RNA extracts that was treated with DNase prior to reverse transcription, in comparison with the undigested ones [1]. The good outcome of the DNase digestion can be then checked by performing an RT-PCR assay in which the reverse transcriptase is not included.
The following protocols describe two different DNase treatments that can be chosen on the basis of RNA availability.
2 DNase Digestion Followed by Phenol/Chloroform Extraction
This protocol allows complete removal of DNase enzyme after DNA digestion [2]. The adjustable volumes of reaction allow digestion of even large amounts of DNA, also when RNA concentration is very low. This protocol is recommended when an RNA solution is not pure (presence of co-extracted contaminants) or when a high RNA quantity should be used in the following analyses. It is not the best choice when the starting amount of RNA is very low (for example, when recovered from small biopsies or after microdissection). In such case, Protocol 18.3 is suggested.
2.1 Reagents
Reagents from specific companies are reported here, but reagents of equal quality purchased from other companies may be used.
All reagents should be RNase freeFootnote 2 and DEPC treated
-
DEPC-treated water (DEPC H 2 O): add 1 ml of diethylpirocarbonate (DEPC) (0.1% final concentration) to 1 l of sterile water at 37°C overnight under fume hood and then autoclaveFootnote 3
-
10 U/μl DNaseI FPLC pure (cod. 27-0514 GE Healthcare)
-
10× Reaction buffer: 400 mM Tris-HCl (pH 7.5), 60 mM MgCl2
-
Phenol/H 2 O
-
Chloroform
-
0.8 M LiCl in absolute EtOH
-
1 mg/ml Glycogen
-
Absolute EtOH
2.2 Equipment
-
Adjustable pipettes,Footnote 4 range: 2–20 μl, 20–200 μl, 100–1,000 μl
-
Nuclease-free aerosol-resistant pipette tips
-
1.5 ml tubes and 0.2 ml tubes (autoclaved)
-
Thermomixer (e.g., Eppendorf)
2.3 Method
-
A 20 μl reaction volume is prepared, adding the following components in a 1.5 ml tube:
10 × Reaction buffer
2 μl
10 U/μl DNaseI
1 μl
Total RNA
2 μgFootnote 5_18
DEPC H2O
to 20 μl finalFootnote 6_18
-
Incubate the mixture at 37°C for 20′. Spin down the condensate from the tube walls.
-
Bring the final volume to 100 μl with DEPC-treated H2O and add 70 μl phenol/H2O + 30 μl chloroform.Footnote 7 Vortex, keep on ice for 20′ and spin the tube for 20′ at 14,000 rpm in a microcentrifuge at 4°C.
-
Carefully transfer the supernatant to a new tube (avoid touching the interphase), and add three volumes of 0.8 M LiCl in absolute EtOH and 5 μl of glycogen solution. Mix the solution by inversion.
-
Leave the solution at –20°C for 24 h.
-
Spin the tube for 20′ at 14,000 rpm in a microcentrifuge at 4°C; remove the supernatant without disturbing the pellet.
-
Wash the pellet with 100 μl ice cold absolute EtOH and spin the tube at 14,000 rpm for 10′ at 4°C.
-
Remove the supernatant, air dry the RNA pellet and resuspend it in the proper amount of DEPC-treated H2O.
-
Determine the new RNA concentration photometrically at 260 and 280 nm, adding 1 μl RNA solution to 199 μl of H2O.Footnote 8
-
The solution is ready to be used for RT-PCR. Alternatively, RNA can be stored at –80°C until use; it is better if it is divided into aliquots to avoid repeated freezing and thawing.
3 DNase Digestion Followed by Heat Inactivation
This protocol can be used when the starting amount of RNA is low and DNase removal by extraction could determine its complete loss [3].
3.1 Reagents
Reagents from specific companies are here reported, but reagents of equal quality purchased from other companies may be used.
All reagents should be RNase freeFootnote 9 and DEPC treated
-
DEPC-treated water (DEPC H 2 O): Add 1 ml of diethylpirocarbonate (DEPC) (0.1% final concentration) to 1 l of sterile water at 37°C overnight and then autoclaveFootnote 10
-
10 U/μl DNaseI FPLC pure (cod. 27-0514 GE Healthcare)
-
10× Reaction buffer: 400 mM Tris-HCl (pH 7.5), 60 mM MgCl2
-
25 mM EDTA Footnote 11
3.2 Equipment
-
Adjustable pipettes,Footnote 12 range: 2–20 μl, 20–200 μl, 100–1,000 μl
-
Nuclease-free aerosol-resistant pipette tips
-
1.5 ml tubes and 0.2 ml tubes (autoclaved)
-
Thermomixer (Eppendorf)
3.3 Method
-
Hereafter, a 20 μl reaction volume is reported, adding the following components in a 1.5 ml tube:
10 × Reaction buffer
2 μl
10 U/μl DNaseI
1 μl
Total RNA
2 μgFootnote 13_18
DEPC H2O
to 20 μl finalFootnote 14_18
-
Incubate the mixture at 25°C for 15′.Footnote 15 Spin the tube to collect the condensate from the tube walls.
-
Add 2 μl of 25 mM EDTA to the solution; mix and heat at 65°C for 10′ to inactivate DNase. Immediately, chill the solution on ice and spin the tube.
-
Determine the RNA concentration photometrically at 260 and 280 nm, adding 1 μl RNA solution to 199 μl of H2O.Footnote 16 Alternatively, for a more approximate quantification, determine the new RNA concentration simply taking into account the starting RNA concentration and the reaction volume before and after DNase treatment.
-
The mixture can be used directly for RT-PCR. Alternatively, RNA can be stored at –80°C until use; it is better if it is divided into aliquots.
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.
References
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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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