I was trying to see the expression of a gene in post-implantation mouse embryos from 9.5-13.5dpc. The preferred technique is obviously whole embryo in-situ hybridization but, I first thought of checking by quantitative PCR. Initially, I used to homogenize the embryos with a tissue homogenizer, TRIZOL. Later, I started homogenizing only with pipetting. The problem is while in the former case, I used to get whopping expression of the gene in12.5-13.5dpc embryos, in the latter case, I got very less significant expression at those stages. Is it because of the variation in the homogenizing procedure or is it because I am using an out-bred strain (CD-1) for my analysis?
Pipetting is not very much effective in extracting RNA from the tissue sample. Hand held battery operated tissue homogenizer
(http://www.thomassci.com/Equipment/Grinders/_/CORDLESS-DRIVE-UNIT-FOR-PELLET-PESTLES?q=Kontes) is the preferred option. The regular microprobe tissue homogenizers are sometimes too harsh and too strong to be controlled in a regulated manner and it produce too much heat during sample prep.
Did yo notice any big difference in the RNA concentration after purification? What was the A260/A280 ratio? Was it around 2 or less than 2?
Hello Amritlal,
Thank you for your response. Actually, the first time I used the homogenizer, I noticed on gel that there was a degradation of the RNA. So, I switched to pipetting. Upon pipietting, actually there isn't much change in the concentration of the RNA and the 260/280 ratio comes like 2.02-2.09. But, the downstream PCR amplification results are hugely variable.
Sometimes the tissue you are working with may have high RNAse activity that potentially degrade purified RNA upon storage or over time. I know cartilage is such kind of tissue. I am not sure about the RNAse activity in the mouse embryo. I believe when you use homogenization based technique lot more RNAse become activated which eventually cause RNA breakdown and you see degradation product in the gel.
As a trial you can add little more RNAse inhibitor while purifying RNA from the tissue.
That will be my best bet.
Evitate heat
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