Consider the extracellular matrix protein Fibulin 1 (see: http://www.genecards.o

Question

Consider the extracellular matrix protein Fibulin 1 (see: http://www.genecards.org for more information), which in humans is encoded by the gene FBLN1. a) Briefly discuss the various methods that mightbe used to examine levels of expression of FBLN1 at the RNA level. What are advantages and disadvantages of each method? b) Describe how you would determine whether you were detecting mRNA as opposed to DNA c) FBLN1 has 4 alternative transcripts (GenBank accession numbers NM 001996, NM_006485, NM_006486, NM _006487). Describe a scheme for distinguishing among these transcripts. d) Briefly discuss the various methods that rmight be used to examine levels of expression of Fibulin 1 at the protei level. What are advantages and disadvantages of each method? e) Consider the commercially available antibody from GeneTex (http://www.genetex.com), catalog number GTX92024. i) Briefly describe how you would use this in a western blot, include the polyacrylamide gel concentration and the secondary antibodies you would use. i) Briefly describe how you would test this antibody to determine whether it would work in immunohistochemistry.

Explanation / Answer

a) There are many ways which can be used to examine the levels of expression of FBLN1 at RNA level:

1. In-situ:

Labeled probe can be hybridized with the RNA and then visualized under a microscope to examine gene expression directly.

disadvantage: Unpractical to test many genes

2 RT- PCR: (Real-Time PCR)

cDNA prepared from the RNA can be quantified using this method.

disadvantage: Technically difficult and expensive.

3. Microarray:

Microarrays are used to find differences in gene expression between two samples. It is used as a comparative method.

disadvantage: technically difficult, require well-studied genome.

b)

In case of In-situ specific probe against the RNA is used, so cross-reaction with DNA is highly improbable. While during RT-PCR the samples are treated with DNase first, then the cDNA is prepared, hence only detection of the RNA transcript level occurs.

c) RNA-seq, CAGE can be used to study different transcripts of the gene.

d) Methods to detects Fibulin 1 at protein level:

1. Western blot:

2. ELISA

3. Immunocytochemistry.

4 LC/MS.

Related Questions

Navigate

• Browse (All)
• Browse C
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.