8. (7 points) Short answers to questions: a. Compare and contrast Northern Blot

Question

8. (7 points) Short answers to questions: a. Compare and contrast Northern Blot and qReal-Time RT PCR techniques in following aspects: Which technique is more sensitive? Which technique displays a direct detection of RNA from the sample? Which technique may produce false positive signal? Why? When using qReal-Time RT PCR, how do we determine exact concentration of our test RNA? b. Reporter genes help us to reveal expression levels and patterns at the protein level. Several GFP transgenic mutant mice were designed: mouse #1 is expressing GFP in the eye tissue mouse #2 is expressing GFP in the skin tissue mouse #3 is expressing GFP in the heart and skin tissue mouse #4 was expressing GFP during embryonic stages only and not expressing GFP post-birth. Describe the general design/composition of the GFP reporter transgenic constructs for each mutant mouse

Explanation / Answer

Northern blot analysis is effective in gene analysis but when compared to q-RT PCR is much more sensitive. q Rt PCR is very challenging in obtaining the results but it permits the comparison of transcript quantities between samples by coamplifying the gene of interest with a housekeeping gene which acts as a control.

Northern blot is the technique used in molecular biology to study gene expression by detection of RNA. The resolution of RNA is better in Northern Blot analysis because a specific RNA is detected as a sharper band by hybridization. The size of the RNA can be determined by the migration of distance of the RNA with those of the markers in the sample added by us and seen in the stained gels of eithidium bromide after electrophoresis.

Both the technique gives the false positive results in spite of taking many precautions in PCR and northern blotting.

Performing PCR in real times means that product formation is measured at each cycle as it is occurring by measuring a change in a florescent reporter signal. with traditional PCR the end point at the PCR is measured only once after the cycling program has ended. In real time PCR the starting template is RNA and is reverse transcribed into a cDNA target prior to PCR.

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