The Huck Institutes of the Life Sciences

Considerations for real-time qPCR sequencing

Things you need to be aware of when planning a real-time qPCR analysis at the Genomics Core Facility

Best practices and recommendations

 

Considerations for each run:

  • Triplicates of each sample.
  • Have a no amplification control (NAC) per RNA sample.
  • DNAse treatment of your RNA during extraction is highly recommended. Our protocol has a second treatment which can further decrease genomic DNA. A Reverse Transcriptase step follows and then an aliquot is removed for the PCR step. No TRIAZOL unless you run a second extraction with another procedure.

 

Housekeeping genes

  • Cyclophilin is usually the best for most systems. Ubiquitin is good for plants and 16s for bacterial. 18S, Ribosomal phosphoprotein (RPPO), and GAPDH can be considered. The reference gene should give constitutive expression and not be affected by treatments. Cts should not vary by more than one Ct if equivalent amounts of nucleic acid are assayed.
  • There is no additional charge to run one reference gene with your samples. We do charge for additional ones.
  • The relative levels of the gene of interest are determined using the delta delta Ct method and the reference gene.

 

Copy number

  • To determine copy number of message, cRNA standards can be run on every plate. 6 to 8 cRNA standards per plate could be run as singlets or these can be run in duplicate as well.
  • cRNA can be prepared from a plasmid containing the cDNA of interest using available kits if you have a T7, T3, or SP6 promoter. Otherwise, it becomes more difficult to prepare the cRNA.
  • Most studies only evaluate relative levels of expression and do not require this tedious technique.

 

Primers

Every sequence requires a set of Forward and Reverse Primers. Please submit the DNA sequence (or cDNA sequence for real-time PCR) so that the appropriate primers and probe can be selected. For real-time PCR it is best to select probes or primers that span introns, however, this is not essential. To select these primers and probes, the location of introns is required.

  • In order to be highly efficient and sensitive, PCR products must be 70 to 100 bases long. Therefore, any primers you are using currently have probably been selected to amplify much larger products and are not usable.
  • The software selects the probe and then the two primers. The probe Tm must be about 68 or 69 degrees and the primers 9 to 10 degrees lower.
  • There are other limitations on each of these which the software is set up to select.
  • You can indicate a particular region to select the primers/probe in and we can check to see if the software will allow that area to be used.
  • You can use Applied Biosystems ready-made assays for genes. Human, mouse and rat are available.

 

Optimization

  • Linearity of the amplification of gene of interest is checked with serial dilutions of the RNA and linearity of the reference gene is checked as well. PCR efficiency should be within 5% between the two genes. A slope of -3.3 is equal to 100% efficiency.
  • Optimization may cost $50. You must also be aware that optimization may fail with the particular set of primers that were made. In this case another set will have to be designed and tried. This is not something we can predict.

 

Probes

  • Every sequence requires a fluorogenic probe. This oligo can be obtained from several companies ($110 and approximately 2000 reactions per probe).

 

SYBR Green vs Taqman

SYBR Green is a non-specific dye which can be used to measure gene expression. Many people want to use SYBR Green so that they won't have to buy the TaqMan Probe. However, SYBR Green is a dye which binds to any double-stranded DNA. Therefore, it will not only bind to your PCR product but to non-specific products that have been amplified as well as primer dimers.  Due to this non-specificity,  SYBR Green requires more time for optimization than TaqMan and non-specificity may be an issue.

 

If you want to use SYBR Green…

You can reserve time on the real-time machines and bring a prepared plate over. The software programs necessary for analysis are available for you to use to analyze your data as well as check melt curves.