Preparation, Quantification, Determination of Quality, and Storage of DNA and RNA

1. Template preparation and quality

Since PCR consists of multiple rounds of enzymatic reactions, it is more sensitive to impurities such as proteins, phenol/chloroform, salts, and EDTA than single-step enzyme-catalyzed reactions. Purity of nucleic acid templates is particularly important for real-time PCR, since contaminants can interfere with fluorescence detection. QIAGEN offers a complete range of sample technologies to ensure the highest-quality templates for PCR, including kits for purification of plasmid DNA, genomic DNA, and viral nucleic acids, and kits for purification of total RNA and mRNA. QIAGEN also offers a range of automated solutions for automated purification of nucleic acids.

2. Determining concentration and purity of nucleic acids

The concentration of DNA and RNA should be determined by measuring the absorbance at 260 nm (A₂₆₀) in a spectrophotometer. For accuracy, absorbance readings at 260 nm should fall between 0.15 and 1.0. Brief guides to spectrophotometric and molar conversion values for different nucleic acid templates are listed in tables "Spectrophotometric conversions for nucleic acid templates" and "Molar conversions for nucleic acid templates".

Note that absorbance measurements cannot discriminate between DNA and RNA. Depending on the method used for template preparation, DNA may be contaminated with RNA, or RNA may be contaminated with DNA, and either of these will result in misleadingly high A₂₆₀ values. It is particularly important to bear this in mind when preparing standards for absolute quantification.

The ratio between the absorbance values at 260 nm and 280 nm gives an estimate of the purity of DNA or RNA. To determine nucleic acid purity, we recommend measuring absorbance in 10 mM Tris·Cl,^‡ pH 7.5. Pure DNA and RNA have A₂₆₀/A₂₈₀ ratios of 1.8-2.0 and 1.9-2.1,^§ respectively. Lower ratios indicate the presence of contaminants such as proteins.

3. Storage of DNA and RNA

Purified RNA should be stored at -20°C or -70°C in RNase-free water. When RNA is purified using QIAGEN kits, no degradation is detectable for at least 1 year under these conditions. Purified DNA should be stored at -20°C or -70°C under slightly basic conditions (e.g., Tris•Cl, pH 8.0) because acidic conditions can cause hydrolysis of DNA. Diluted solutions of nucleic acids (e.g., dilution series used as standards) should be stored in aliquots and thawed once only. We recommend storage of aliquots in siliconized tubes where possible. This avoids adsorption of nucleic acids to the tube walls, which would reduce the concentration of nucleic acids in solution. For long-term storage of DNA samples, we recommend using QIAsafe DNA Tubes and 96-Well Plates, which provide stable, room-temperature storage.

4. RT-PCR

When performing two-step RT-PCR using the QuantiFast SYBR Green PCR Kit, QuantiFast Probe PCR Kits, QuantiTect SYBR Green PCR Kit, QuantiTect Probe PCR Kit, or QuantiTect Multiplex PCR Kits, the RNA must first be reverse transcribed into cDNA in an RT reaction. Failure of the subsequent PCR is often a result of the limitations of the RT reaction. On average, only 10-30% of template RNA molecules are reverse transcribed into cDNA. The expression level of the target RNA molecules and the relatively low efficiency of the RT reaction must be considered when calculating the appropriate amount of starting template for subsequent PCR. However, adding high volumes of the RT reaction to the PCR can affect C_T values. Generally, RT volumes up to 10% of the total PCR volume do not affect results.

Total RNA or messenger RNA (mRNA) can be used as template in RT reactions. We recommend RNeasy Kits for purification of total RNA and Oligotex Kits or TurboCapture mRNA Kits for purification of mRNA. For reverse transcription, we recommend the QuantiTect Reverse Transcription Kit. The kit provides removal of genomic DNA contamination and includes a primer mix for cDNA synthesis from all regions of mRNA transcripts.

^‡When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs), available from the product supplier.
^§Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris•Cl, pH 7.5) with some spectrophotometers.