Lab 05 - Quantitative Detection Of Gene Expression Part I: RNA Extraction, Evaluation and Reverse Transcription Flashcards
What were the objectives of this Lab?
1) Extract total RNA
2) Determine the quantity of RNA using Spectroscopy
3) Determine RNA quality using gel electrophoresis
4) Reverse transcribe RNA into cDNA
How did we use spectroscopy to determine the quantity of the RNA?
We used the OD260 (RNA) and OD280 (impurities) reading, as this will tell us how much light was able to pass through the sample. We then used this number with our equation to determine the concentration.
[RNA] = 40 μg / mL × OD260 × dilution factor
How did we use gel electrophoresis to determine RNA quality?
When RNA shows up on a gel, if the quality is good, there should be two distinct bands with little clouding.
What is an operon (prokaryotic only)?
An operon occurs when multiple genes with similar protein products are stacked under one promoter and are all transcribed together.
What is a polycistronic mRNA?
The product of an operon which contains the transcribed products of many genes.
What may the coding regions of eukaryotic genes (exon) be interrupted by?
Introns: Non-coding regions
How are introns removed?
Post transcriptional processing
How is the Poly-AAA tail used? How can we take advantage of this?
To purify and and separate mRNA from other RNA. We can use this as a primer binding site using a Poly-TTT primer for reverse transcription.
What are the differences between using random hexamer primers in reverse transcription as opposed to poly-TTT primers?
To use hexamers we must know the genetic code of the RNA we are looking at. However, when using a poly-TTT primer, we can only target mRNAs and all of them will be reverse transcribed so we must find a way to isolate our gene of interest.
How do you convert RNA to cDNA?
Use a reverse transcriptase (MMLV Reverse Transcriptase).
Why may looking at reverse transcribed genes be more helpful than looking at the original DNA / genome?
We can find the specific genes expressed and in their specific quantities in that tissue or cell, rather than all genes expressed in that organism.
Which gene did we look at and where was it expressed in higher quantities?
We looked at LinS or Linalool in lavender plants, which is expressed more in the flowers than the leaves.
Why did we use actin?
Actin is a housekeeping gene, and as such is present in all cells, this allows us to ensure that our procedure occurred properly and the results of the LinS are most likely correct.
What are the different methods which have been developed to detect the expression of a particular gene?
Northern Blotting / PCR (mRNA)
Western Blotting / SDS Page (protein)
Enzyme Assay (Gene function not expression)
What is Standard RT-PCR?
End point analysis of PCR products
