Question

You are planning to perform LCM to isolate cells from complex tissue to obtain characteristic gene expressions by RNA analysis:

a. Is there any damage to the genetic material from the LCM process?

b. Is it necessary to dehydrate the samples?

c. Is it necessary to stain the slide for LCM?

d. What is the minimum number of cells necessary for molecular analysis?

Answer 1

LCM - Lase Capture Microdissection

a. Is there any damage to genetic material from the LCM process?

There is no damage to the genetic material from the LCM process - neither structure not its chemistry is affected that is why it is a popular method for isolating cells from complex tissue.

b. Is it necessary to dehydrate samples?

Proper dehydration of the sample is necessary after staining, as it prevents the upward adhesive force between the slide and the tissue, and prevents the difficulty of separating the tissue from the slide. The samples which are still having some moisture residual will make the tissue translucent compared to dehydrated ones.

c. Is it necessary to stain the slide for LCM?

Tissue staining actually helps in the guiding process while dissecting and to select specific cells during LCM.

Hematoxylin and Eosin stains are used for guiding dissection so they do not inhibit the analyse nor lead to protein losses seen with other stains.

Cresyl violet is used in RNA extraction effectively with good quality RNA.

But most histological stains affects protein separation and so now the introduction of navigated LCM.

With mRNA extraction immuno staining which was fluorescent and enzymatic detection based were working better.

d. What is the minimum number of cells necessary for molecular analysis?

Pilot tests should be run to find out the minimum number of cells necessary for molecular analysis. So it is better to start with the high number like 10000 cells and then scale down to minimum sample size based on the quality, quantity of the amount of material extracted and the integrity of the biomolecule.