RNA sample may be of poor quality
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).
How to identify PCR inhibition
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 70–80% of protein in the samples—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 on a sample using a new purification method—choose your RNA extraction kit based on sample type. Refer to the RNA Isolation Decision Tree to help make the correct 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 using a lower template concentration at which it is known that PCR inhibition does not affect the real-time PCR results.
Your samples contain degraded RNA
RNAs are very sensitive to degradation. Although some applications are tolerant to partially degraded RNA (e.g., RT-PCR), it is still important to assess RNA integrity to ensure that samples being compared are of similar integrity.
How to assess RNA integrity:
Denaturing Agarose Gel Electrophoresis: For total RNA samples, discrete, thick 28S and 18S ribosomal RNA (rRNA) gel bands at an approximate mass ratio of 2:1 are indications of high integrity (below, lane 3). Smeared 28S and 18S rRNA bands and a 28S:18S rRNA ratio less than 2:1 indicate RNA degradation (lane 2). Note that some tissues and species yield total RNA with a 28S:18S rRNA ratio of less than 2:1. For messenger RNA or small RNAs use a northern blot to assess integrity if you use this technique.
Intact vs. Degraded RNA.
Degraded total RNA and intact total RNA (2 µg each) were separated alongside the Ambion® RNA Millennium Markers™ on a 1.5% denaturing agarose gel. The 18S and 28S ribosomal RNA bands are clearly visible in the intact RNA sample (lane 3). The degraded RNA appears as a lower molecular weight smear (lane 2).
Agilent® 2100 Bioanalyzer: This instrument uses capillary electrophoresis (microfluidics) to analyze nucleic acids. It requires only 25 ng of RNA input. A 2:1 ratio in the area under the peaks for 28S and 18S rRNA indicates intact total RNA (see figure below). Degradation is indicated by less pronounced peaks for 28S and 18S rRNA and a 28S:18S rRNA ratio significantly less than 2:1.
Agilent® 2100 Bioanalyzer Scans of High Integrity Total RNA. The 18S and 28S peaks are clearly visible at 39 and 46 seconds, respectively
Follow these recommendations to obtain high quality RNA and improve the success of downstream applications:
- Store samples carefully prior to RNA isolation.
- Disrupt samples completely to prevent RNA degradation and to increase RNA yield.
- Choose an appropriate RNA isolation kit. Use the RNA Isolation Decision Tree to help make the correct choice.
- Store RNA in RNase-free solutions.
- Accurately quantitate RNA and confirm that RNA is of high integrity and purity.
- Maintain your laboratory workspace free of RNase contamination.
For more information on RNA handling and RNA isolation, refer to: The Basics: RNA Isolation.