RNA that contains inhibitory compounds (e.g., sample preparation reagents, excessive protein) can lead to partial or complete inhibition of downstream PCR. PCR inhibitors originating from the starting material include heparin (>0.15mg/mL), proteins such as hemoglobin (>1mg/mL), polysaccharides, chlorophylls, melanin, humic acids, etc. Contaminants from the nucleic acid extraction phase include SDS (>0.01% w/v), phenol (>0.2% w/v), ethanol (>1%), proteinase K, guanidinium, and sodium acetate (>5mM).
Analyze RNA samples with a UV spectrophotometer, bioanalyzer or Nanodrop to assess quantity and quality. A high quality RNA sample should have an A260/A280 UV spectrophotometer reading close to 2. It has been observed that an A260/A280 reading of 1.8 suggests there is about 70&endash;80% of protein in the samples&emdash;there are many proteins that inhibit both PCR and reverse transcription.
Inhibition plot: You can use real-time PCR data from standard curve plots to determine whether inhibition is occurring at a level that causes spurious results. When used to characterize inhibition, these semi-log standard curves are referred to as inhibition plots. Please refer to the RNA Preparation and Reverse Transcription section of the Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR.
Perform RNA purification process on a sample using a new purification method. Choose your RNA extraction kit based on sample type. Refer to the RNA isolation kit decision tree to help make the right reagent/kit choice.
Further purify your samples: RNA with a significantly lower A260/A280 ratio should be further purified by phenol-chloroform extraction, LiCl precipitation, or washing to remove residual salt.
Test your sample at a lower template concentration at which it is known that PCR inhibition does not affect the real-time PCR results.