I´ve checked the primers 36B4 in UCSC browser PCR in silico, and found that they hibridize with sequences in 3 chomosomes. So it could not be considered as inuque copy, is it right??
Somebody can help me about it?
36B4 really is a unique and highly conserved gene. I think you just needed to use more stringent or optimized primer binding conditions ( or maybe you caught pseudogenes )
https://www.uniprot.org/uniprot/Q0H9W3
http://blog.biosearchtech.com/TheBiosearchTechBlog/bid/27718/The-New-Favorite-Reference-Gene-36B4-a-k-a-RPLP0
See how it has high similarity to other proteins but is unique by itself. Note that it also has 8 related pseudogenes on chromosomes 1,2,3,11,14,15,18 of the human genome.
Also see this article on the stability and optimal primer melting temperatures of several housekeeping genes. 36B4 seems to melt best at 83 degrees (Fig 1-C) Article Selection of Suitable Reference Genes for Quantitative Real-...
hi! If you want to check this by yourself there is a quite interesting and effective way.
1. DNA purification from human cell line or WBC fraction. For both variant 100 000 - 1 000 000 cells.
2. Measurement of DNA concentration with Promega Quantus or Thermofisher Qubit (fluorescence).
3. Counting of copies depending on concentration. How many genomes could be in 1 ul of your DNA sample. It depends on concentration.
4. SYBR based PCR with a serial dilutions of your DNA sample with known concentration. Usually I use 5-fold dilution. 4 steps is enough. As a result you able to determinr reaction effectiveness.
5. Next you have to compare theoretical and practical results. For example you determine that in 1 ul of your DNA you have 50 000 copies of human genome (I think less, but for example). After PCR you receive (on the base on standard curve) 100 000. In this case you have 2 copies of your sequence in genome. If you receive approximatly 50 000 - great. Only one copy. )
Good luck.
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