FAQs on Real-Time RT-PCR

 

Why PrimerBank?

PrimerBank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification (real-time PCR). There are several ways to search for primers: by GenBank Accession, NCBI protein accession, LocusLink ID, PrimerBank ID or Keyword (gene description). PrimerBank contains about 180,000 primers covering most known human and mouse genes.

Polymerase Chain Reaction Amplification (PCR) is one of the most actively used techniques in molecular biology. In recent years, PCR has been increasingly used for gene expression detection or quantification. It is a more convenient method in gene expression studies comparing to other techniques, such as Northern Blot. One common problem in PCR is the non-specific amplifications of other gene products because cDNAs libraries of thousand of genes are often used as PCR templates. Therefore, we need to carefully design PCR primers that specifically amplify the genes of interest. Unfortunately, most available primer design programs only focus on primer chemical properties, such as melting temperature, GC content, secondary structure, etc.  Little emphasis is given to primer mispriming to other genes. In contrast, all primers in PrimerBank were carefully designed to ensure gene specificity. 

 

How about the quality of these primers?

The primer selection algorithm has been extensively tested by real-time PCR experiments for PCR specificity and efficiency. Up to now, we have tested over a thousand primers and the design success rate is > 99%.

 

How to search PrimerBank by gene IDs?

You may search PrimerBank by GenBank Accession, NCBI protein accession, LocusLink ID, or PrimerBank ID.

 

How to search PrimerBank by keywords (gene description)?

Search terms are automatically combined if they are separated by space. For example, search "kinase s6" returns all records with both keywords "kinase" AND "s6". AND and OR Boolean operators may also be used. Here are a few examples:
 

Keyword	Interpretation
kinase s6	Searching for both kinase and s6
kinase and s6	Searching for both kinase and s6
Cytochrome or cyp	Searching for either Cytochrome or cyp

Currently, only a limited set of keywords (keywords defined in the protein definitions) can be searched. For example, you cannot search by gene symbols. If you did not find your genes, please look up the corresponding gene IDs (DNA accessions, proteins accessions, or LocusLink IDs) and search by IDs.

 

Why did the retrieved records sometimes have different IDs than originally submitted?

Because of the sequence redundancy in GenBank, each gene is usually represented by more than one NCBI record. PrimerBank sometimes uses the NCBI LocusLink index file to map multiple sequence records to the same gene locus. As a result, a different accession number other than originally submitted may be retrieved. However, both accessions represent the same gene. If a retrieved record has a different GenBank accession, a LocuLink ID will appear in the gene description field.

 

How can I save my results?

You may use your browser's save function to save the web page in your local computer. Other saving options will be implemented later.

 

How were the primers designed?

All the primers in PrimerBank were designed using a program called uPrimer. Great care has been given to avoid primer mispriming to other known genes in a genome. Here is a list of criteria for gene specific primer design:

1. The primer length range: 19 - 23 nt, with the optimal length at 21 nt.
2. The primer GC percentage range: 35% - 65%.
3. The delta G value for the five 3’ end-bases is at least -9 kcal/mol.
4. The primer Tm range: 60 - 63 °C, determined by the Nearest Neighbor Method.
5. The PCR product length range is 100 - 250 bp. If this requirement cannot be satisfied, alternative ranges will be used.
6. The default number of primer pairs designed for each sequence is 3.
7. No primer is designed from low-complexity regions.
8. A primer does not contain 6 or more contiguous same nucleotides.
9. A primer does not contain any ambiguous nucleotide.
10. No repetitive 15-mer from other gene sequences in the genome (for both strands) anywhere in a primer.
11. No repetitive 13-mer from non-coding RNA sequences (for both strands) anywhere in a primer.
12. The global BLAST score for any primer is less than 30 (equivalent to 15-mer perfect match).
13. The maximum Tm for the 3’ end perfect match to other gene sequences does not exceed 46 °C; does not exceed 42 °C when compared to non-coding RNA sequences (Tm determined by the Nearest Neighbor Method).
14. For primer secondary structure (the primer-primer self-annealing)
    1. No repetitive 5-mer is allowed anywhere when a primer sequence is compared to its complementary strand.
    2. The four 3’-end bases should be unique when compared to the primer’s complementary strand.
15. The forward and reverse primers should not anneal to each other. The filter setting is the same as in the primer secondary structure filter.
16. For sequence secondary structure (the primer-sequence self-annealing)
    1. No repetitive 9-mer is allowed when a primer sequence is compared to the complementary strand of its cognate sequence.
    2. The BLAST score is less than 18 when a primer sequence is compared to the complementary strand of its cognate sequence.

 

What annealing temperature should be used in PCR experiments?

All the primers in PrimerBank have melting temperatures (Tm) of 60 - 63 °C. A higher annealing temperature results in more specific priming. Previous studies indicated sufficient priming should occur at primer Tm. Therefore, an annealing temperature of 60 °C is recommended for all PrimerBank primers. Non-specific PCR products are likely to occur at lower annealing temperature. We have tested a few hundred primers under 60 °C annealing temperature and the PCR experiments worked very well.

 

How were the primer melting temperatures calculated?

The primer Tm values are calculated using the Nearest Neighbor Method with the up-to-date thermodynamic parameters. Tm values are also dependent on primer and salt concentrations. Higher concentrations of primer and salt usually lead to higher Tm. The Tm values included in PrimerBank are for 0.25 uM primer, 1.5 mM Mg²⁺ , 50 mM Na⁺ , and 0.8 mM dNTP, which are typically used in PCR experiments. Other common PCR conditions only affect the Tm slightly.

 

What is the best amplicon size?

To guarantee PCR efficiency, small PCR products are recommended. Most PrimerBank primers lead to amplicons in the size range of 100 - 250 bp. PCR efficiency is close to 100% in this range (supported by real-time PCR experiments). However, PCR efficiency may be reduced for much larger PCR products. . Less than 1% of PrimerBank primers lead to >400 bp amplicons. Please be cautious about PCR efficiency when these primers have to be used.

 

Do the primer pairs span introns?

Sometimes it is desirable to select a primer pair that spans intron. In this way, genomic DNA contamination can be closely monitored. Usually a PrimerBank primer pair spans intron because a typical exon is quite small. The primer intron-spanning information will be included in the next version update.  If it is important for you to be sure that a primer pair spans intron, you may simply do a BLAST search of the primer pair sequences, or better the amplicon sequences (listed in the primer detail page) against the genomic sequences.

 

I see multiple bands on gel or multiple peaks in the melting curve. Are these non-specific?

Agarose gel electrophoresis or melting curve analysis may not always reliably reflect PCR specificity. From our experience, bimodal melting curves are occasionally observed for long amplicons even when the PCRs are specific. The observed heterogeneity in melting temperature was due to internal sequence inhomogeneity (e.g. independently melting blocks of high and low GC content) rather than amplicon contamination. On the other hand, for short amplicons very weak bands migrating ahead of the major specific bands are occasionally observed on agarose gel. These weak bands are super-structured or single-stranded version of the specific amplicons in equilibrium state. Although gel electrophoresis or melting curve analysis alone may not be 100% reliable, the combination of both can always reveal PCR specificity in our experience.

 

I still see non-specific PCR bands. What should I do?

Although all PrimerBank primers are designed to be gene specific, we still need to be very careful about PCR conditions. 

Non-specific primer extension of only a few bases at low temperature by DNA polymerase can easily lead to non-specific PCR amplifications. Therefore, hot-start PCR is STRONGLY recommended. In fact, I only do hot-start PCR in my experiments. I routinely use AmpliTaq Gold polymerase (Applied Biosystems) for hot-start PCR.

The annealing temperature may affect PCR specificity. To avoid non-specific PCR products, a high annealing temperature (the smaller one of the two Tm values from the primer pair) and a short annealing time are recommended.

If you still see non-specific bands, it could mean the primer pair in use is the problem. Although great care has been given to design PrimerBank primers, the success rate is not 100%. Less than 1% of the primers may have design problems (see our paper for detailed discussion). In this case, please try a different primer pair for the same gene. Click here if you would like to report primer problems.

 

There is no PCR product. What should I do?

Poor quality of PCR templates, primers, or reagents may lead to PCR failures. First, please include appropriate controls to eliminate these possibilities.

Some genes are expressed only in certain tissues. Please first read literature to make sure your genes are included in the cDNA templates. In our experience, this is the most likely cause for negative PCR results. If you are sure the genes are expressed, then try lowering the annealing temperature (to Tm - 5 °C) and increasing the annealing time to ensure sufficient primer annealing.

If you still could not see any PCR band, it could mean the primer pair in use is the problem. Although great care has been given to design PrimerBank primers, the success rate is not 100%. Less than 1% of the primers may have design problems (see our paper for detailed discussion). In this case, please try a different primer pair for the same gene. Click here if you would like to report primer problems.