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Considerations

Things you need to be aware of when planning a real-time PCR analysis at the University Park Nucleic Acid 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

  • 18S, Ribosomal phosphoprotein (RPPO), GAPDH, cyclophilin. Or possibly a gene of your choice. 
  • The housekeeping gen can sometimes be multiplexed or run in the same tube as an internal standard. There will be no additional cost for these primers and probe. There is no guarantee that the housekeeping gene and your gene of interest can be optimized to multiplex.
  • The relative levels of the gene are determined in relation to the housekeeping gene using an Excel spreadsheet and a formula and derivation available from us.

Copy number

  • If you want 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

  • Each sequence requires optimization of Mg2+, primer concentration and probe concentration. For real-time PCR an appropriate positive RNA sample should be submitted. This must be a positive sample from the tissue or cell type you will be using.
  • Linearity of the protocol is checked with serial dilutions of the RNA and linearity of the housekeeping gene is checked as well.
  • Optimization may cost $50 to $100. 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 ($150 and approximately 2000 reactions per probe).