You may not have selected the correct TaqMan® assay
Applied Biosystems offers more than one million TaqMan® Gene Expression Assays. All assays have been designed with the Applied Biosystems validated bioinformatics pipeline and can be run with the same PCR protocol, eliminating the need for primer design or PCR optimization. However, it is important to select the correct assay for the transcript and RNA source. The Online Ordering Guide and the Online Selection Guide are documents that help you understand all the useful information on the Applied Biosystems homepage that supports the selection of the ideal assay for the intended research.
To ensure that your TaqMan® Gene Expression Assay does not contribute to failure of amplification, you may need to confirm the following information:
- Where is the TaqMan® Gene Expression Assay located?
- Are there any transcript variants for your gene of interest?
- Are you validating microarray experiments with real-time PCR?
You may have problems with reverse transcription
Reverse transcription (RT) is the process by which RNA is used as a template to synthesize cDNA. The single stranded cDNA molecule serves as a template for the real-time PCR. Different RT methods can have significant impact on real-time results. The RT reaction is often overlooked when troubleshooting late CT values (>35) or when no amplification is observed. Considerations have to be made for the type of RT reaction that was performed (one-step or two-step), the linearity of the RT reaction, and the range of RNA input (capacity) the RT chemistry will tolerate.
Your samples contain PCR inhibitors
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, and humic acids. 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.
You may not have enough template sequence
Your gene of interest may not be expressed at a high level in your sample type, or the sample types you extract from yield very low nucleic acid, e.g., obtained by cell sorting, LCM, or FFPE.
For guidance on how to assess the amount of RNA input for your real-time PCR study, refer to page 25 of the Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR.
- Increase the amount of template you use in the real-time PCR reaction.
- Select an RNA isolation kit based on your sample type to maximize yield.
- Preamplify your target sequences: The TaqMan® PreAmp Master Mix Kit is intended for use with very low amounts of cDNA. Up to 100 targets can be simultaneously preamplified in one multiplex reaction. Confirm that all amplicons are amplified uniformly without bias before performing preamplification with limited amounts of samples. See the TaqMan® PreAmp Master Mix Kit Protocol for further details.
Publication: Noutsias, M. et al. (2008) Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 9(1):3
You may have problems setting up the reaction conditions on your real-time PCR instrument
The following thermal conditions are set as the default for the standard mode for all Applied Biosystems real-time PCR instruments:
- 50°C: 2 min
- 95°C: 10 min
- 95°C: 15 sec, and 60°C: 60 sec for 40 cycles
TaqMan® Gene Expression Assays and Custom TaqMan® Gene Expression Assays are designed to be used with the default thermal cycler conditions shown above. If you design TaqMan® primers and probes with Primer Express™ software without changing any design parameters, you can use these default thermal conditions.
Some commonly seen setup errors are:
- Insufficient heat activation of the polymerase in master mixes
- Incorrect setup of the reporter dye
- Incorrect setup of the data collection stage
Where is the TaqMan® Gene Expression Assay located?
- Choose an assay that is located in the coding sequence of the mRNA sequence.
- After Assay search, click on the Assay ID (e.g., Hs01075862_m1, shown below) on the Results page:
- Look at Assay Details in the next window. If the assay was designed in the 5′-UTR that information can be found under Additional Information as shown below:
Are there any transcript variants for your gene of interest?
Which transcript variants of your gene of interest are expressed in the particular tissue/cells used for RNA extraction?
To get more information about variants, do the following:
Once you get the results from your TaqMan® Gene Expression Assay search on the Assay Search results page, use the Alignment Map link to get an overview of all known transcripts for the gene of interest. Hyperlinks from the assay details page direct you to NCBI, where you can access more information about the gene of interest, such as mRNA sequence information, Gene ontology, and GEO (gene expression omnibus) profiles. If you are interested in detecting all transcript variants with your assay, make sure that you select an assay that detects all or most variants.
Use the GeneAssist™ siRNA Workflow Builder to simplify your search for the ideal assay. This collection of over 350 interactive cell signaling pathways gives you easy access to gene and disease information and enables you to order corresponding TaqMan® Gene Expression Assays and Ambion® Silencer® and Silencer® Select siRNAs. The GeneAssist™ Pathway Atlas provides you with lists of genes, proteins and products related to a particular pathway (e.g., BRCA1 pathway), visualized in over 350 interactive pathway maps. You can use the gene lists to configure your TaqMan® Array or TaqMan® Express Plate by exporting it for upload into the respective ordering tools.
Are you validating microarray experiments with real-time PCR?
- If you are performing microarray validation experiments, make sure the assay detects a transcript that matches the array probe or target.
- Use the UMapIt Microarray-to-TaqMan® Assays Mapping Tool to quickly find and order the appropriate TaqMan® Gene Expression Assays to validate microarray results. The UMapIt tool is compatible with whole genome array data from all major microarray platforms.
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
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 Research Use Only. Not for use in diagnostic procedures.