You are interested in studying the expression of the Abcal gene in mice at the R

Question

You are interested in studying the expression of the Abcal gene in mice at the RNA level. In order to study Abcal you isolate total RNA from the following tissues and perform a Northern blot. You probe first for Abcal. After you have results from that experiment (upper box), you strip the probe off the membrane and reprobe for GAPDH. Those results are shown in the lower 1 23 4 5 67 7000 nt Abca1 Gapdh 1400 nt Figure 1. Expression Analysis of Mouse Abc1 by Northern Blotting. Lanes 1-7 represent total RNA isolated from the following specific tissues in the adult mouse:1)Liver 2)Spleen 3)Large Intestine 4)Lung S)Heart 6)Brain 7)Thymus.. A. GAPDH stands for Glyceraldehyde 3-phosphate dehydrogenase, which is an enzyme expressed at a high level in all cells (it is needed for general cell maintenance). Why did you do a subsequent probing for GADPH? B.Which tissues express Abcal? Which has the highest expression? What is shown on the gel that allows you to conclude this? C. Describe (in general terms) the probe used in the Northern blot for Abcal. D. What tissues do not contain Abac1? Explain what leads you to that conclusion. E. You want to determine the subcellular localization of the ABCA1 protein in cells of the thymus using antibodies, briefly how would you do this?

Explanation / Answer

A. GAPDH is required by the cells for its maintenance, so it will always be expressed within all the types of cells. But gene-of-interest can either be expressed or not expressed. If after blotting there is absence of the concerned RNA then it might be because of no expression or the RNA isolation was not proper. Since GAPDH will always be present within the cell, absence of GAPDH shows that RNA isolation or the blotting experiment was not successful. Hence GAPDH helps in keeping our experiment in check. Also, GAPDH is a reference point for quantification of expression levels. Increase in the exposure time increases the thickness of the band. So, if the band of GAPDH is equal in all the cases, then the concerned gene can be compared.

B. According to the figure 1, liver, large intestine, brain, and thymus express Abca1, because the band is observed in these cases and in other cases no band is observed. Since all the GAPDH bands have same thickness, the Abca1 bands can be compared with each other. Upon comparison, Abca1 band in case of thymus tissue is thickest. This shows that thymus tissue has highest expression of Abca1 gene.

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