I have a parasite sample (mixed with host blood) and I need to check gene expressions of parasite using relative quantification (RT_qPCR). For this, I need a good housekeeping gene. I chose 10 genes (suggested as housekeeping) that I need to validate and after choose the most stable one(s). My problem is that my parasite cDNA is mixed with host cDNA and it is impossible to separate them or count the parasite. I do not know how I could choose the best housekeeping gene in this case? I do not have the real amount of parasite cDNA used for qPCR, and I do not have any good internal control. Basically, it is circular problem: In order to validate my target gene I need a good housekeeping gene for internal control, but I do not have an internal control to make relative quantification of my housekeeping genes and to chose the best one. Could you please suggest any study or method that deals with such a case?

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    $\begingroup$ Is the parasite genome sequenced? If so, you may be able to identify a gene that is different enough from host to parasite to design parasite specific primers? $\endgroup$ – akaDrHouse Mar 31 '17 at 13:22
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    $\begingroup$ Your qRT-PCR primers should be specific to one species or another in at least one of the housekeeping genes you choose, it's unlikely all 10 primer pairs work on both species. $\endgroup$ – Artem Mar 31 '17 at 22:11

Two possibilities:

  1. Design primers based on differences in the sequences of the gene for the same protein between the two organisms. (Suggested by @Artem)

  2. Identify an aspect of the metabolism of your parasite that is not present in the host, then use enzymes from that pathway. If the parasite is a worm and the host a mammal, for example, you should surely be able to find something.

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    $\begingroup$ We have primers that distinguish mouse and human Beta-Actin and GAPDH for engraftment experiments. It's not difficult to simply design primers to target one species and not another. $\endgroup$ – Artem Mar 31 '17 at 22:12
  • $\begingroup$ @Artem — Don't tell me, tell the poster in an answer. Only comment on my answer if you think there is a flaw in it, not to point out an alternative. $\endgroup$ – David Apr 1 '17 at 7:34
  • $\begingroup$ Because comments are not answers, and SE Biology has a specific question and answer model I have incorporated Artem's comment into my answer. This way it will be useful for anyone else with the same question. (I do not need the points that will accrue when it is marked up or accepted, which should now be done, I think.) $\endgroup$ – David Apr 5 '17 at 12:35

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