eLa 5.1 Measuring Gene Expression Flashcards
What is the process of Northern Blot hybridization?
In this procedure, RNA from a tissue is isolated, separated by size on a gel and then transferred to filter paper. A radioactive DNA molecule containing the target gene’s nucleotide sequence is then hybridized to the RNA on the filter. When exposed to X-ray film “bands” appear where the probe has bound. The intensity of the band is proportional to the amount of RNA in the sample. If a gene makes multiple mRNAs (alternative splicing) then multiple bands in a given lane will be detected.
What is the process of RT-PCR?
This technical approach employs two steps. The first, reverse-transcription, converts the RNA sample into complementary DNA (cDNA) by the use of the retroviral enzyme reverse transcriptase. Then, the presence of the gene of interest (GOI) is determined by employing PCR, using primers designed to specifically amplify the GOI.
When is it beneficial to use RT-PCR over Norther Blot and vice versa?
RT-PCR - when there is a very small amount of RNA
Northern Blots - when trying to detect multiple forms of splicing
What is gene expression profiling?
Compares the expression of the entire genome between two or more samples. That is, one can catalog which genes are expressed and how much, between normal and disease state.
What type of situation would need gene expression profiling?
An example of the available data a study examining gene expression proto- in normal-appearing colonic mucosa of early onset colorectal cancer (CRC) patients compared to healthy control individuals; the data for the c-fos proto-oncogene can be seen here. The data from this study identified genes (among them, c-fos) that may contribute to early onset CRC susceptibility [5]. The findings have therapeutic, prophylatic and diagnostic implications.
What is one of the key limits of gene expression profiling?
Importantly, gene expression profiling using microarray only measures the expression of mRNAs, and does not differentiate between mRNAs that will produce functional or non-functional proteins.
DNA sequence data is currently routinely obtainable as clinical laboratory data?
Sequence information about specific, clinically relevant genes
You hear about a new diagnostic tool whereby melanoma (a solid tumor) can be diagnosed from a blood sample. The procedure detects the expression of a melanoma-specific gene in the ~10 melanoma cells typically present in a 10 milliliter blood sample. Which of the following technical approaches must the procedure employ?
qRT-PCR
You have obtained tumor samples from a patient with small cell lung cancer. Some of the tumors were removed from the lung, and others are metastases from the liver. You are interested in determining differences in gene expression that contribute to the two tumor phenotypes. Which of the following techniques would be appropriate for your planned study?
Gene expression profiling
You now have a list of over 50 genes that are either up-regulated or down-regulated in the primary tumor as compared to the metastasized tumor. To confirm that these 50 genes are differentially expressed in some fresh tumor samples you have obtained, and to quantify the changes in expression, which technical approach would you employ?
qRT-PCR
You read in a journal that an alternatively spliced mRNA from the NF-kB gene is expressed in alveolar macrophages (macrophages in the lung) of patients with asthma, compared with individuals with no disease. Figure 1 of the paper has the data. What type of data are displayed in figure 1?
A Northern Blot
