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Which gene do we use as a positive control for PCR due to its presence in...

Which gene do we use as a positive control for PCR due to its presence in both granulocytes and monocytes, and its function as a "housekeeping" gene?

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Expert Solution

Positive controls are taken to always produce positive results during experiments. Result of positive control makes sure that we have not committed any procedural error during setting of reaction mixture. For example- We may miss adding dNTPs or mixing Taq DNA polymerase. If we find negative result in positive control then, we can be sure that some fault either in mixture preparation or in setting of PCR reaction has occurred.

We select genes as positive control during any experiments like PCR of DNA of monocytes or granulocytes, Positive control should be present in both the cell types (monocytes and granulocytes). The expression pattern should not fluctuate drastically during any experiment.

Most common house keeping control gene selected for PCR of DNA of monocytes or granulocytes is Beta actin (cytoskeletal protein). Beta actin gene plays a key role in cellular integrity and cell motility. Beta actins are ubiquitously expressed at constant level in all the cells ( including monocytes or granulocytes) unlike other isoforms of actin.

Following 2 factors make it best for use as positive control in granulocytes and monocytes-

-Ubiquitous expression

- It maintains basal level of expression during different situations (treatments).

Maintenance of a basal expression makes it better choice over glyceraldehyde 3 phosphate dehydrogenase (GAPDH) during Reverse transcriptase or real time PCR. Because expression pattern of GAPDH fluctuates in different tissues.

gladiator answered 1 year ago
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