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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.