Flashcards in Lab 05 - Quantitative Detection Of Gene Expression Part I: RNA Extraction, Evaluation and Reverse Transcription Deck (36):
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
What is Quantitative Real-Time PCR?
Examines PCR products during PCR and at end point
What are all the steps of a microarray?
1) Create Probes (Know Gene of Interest)
2) Isolate and Purify RNA
3) Reverse Transcribe RNA into cDNA with different colored labels for different treatments
4) Hybridize labelled cDNA to the chip and wash to remove unbound genes
5) Take photo under red and green laser light
6) Overlap photos to compare expression of genes in different treatments
What is PCR mainly used for?
Replication of DNA using target specific primers and a DNA polymerase enzyme.
What must we do to ensure RNA does not degrade in our sample?
Use enzymes to degrade RNAses and keep our sample on ice to prevent unstable RNA from self break down
What must be ensured as you move tissue to the RLT buffer?
That the tissue does not thaw (liquid N2) before you get it into the buffer to keep RNA intact.
How do we ensure RNA did not degrade during the purification procedure?
Running the RNA sample on an agarose gel electrophoresis and observing two strong bands (18s and 28s rRNA in 1:2 ratio).
What is the charge of RNA? Which side of the agarose will it run to?
Negative to Positive
What is used in the agarose to aid in visualization?
SYBRsafe and loading dye
What is in the loading dye that aids RNA in settling into the wells?
What are the smears seen on agarose gel when testing RNA integrity?
Degraded RNA (smaller) and because it will travels faster, these smears will be seen towards the end of the wells (positive side).
What is the OD260/OD280 ratio of pure RNA?
What are the components of a PCR reaction?
Buffer, Template (RNA, DNA), Primers (hexamers, Poly-TTT), dNTPs (A, T, C, G) and a DNA Polymerase (Taq)
What is the template for a Reverse Transcriptase?
What are some common RNAse degrading or removing compounds used in labs?
'RNase away' and 'RNase out'
Why is knowing the concentration of RNA in our sample important?
Need exact amounts of RNA to add to PCR so the reaction works.
When OD260 = 1.0, what is RNA concentration?
Why is it important to have low impurities in our RNA sample?
Impurities impede the enzymatic reactions
What are the two reasons we reverse transcribe to cDNA?
cDNA is more stable for storage and cDNA can be amplified by PCR (Bonus: cDNA can be inserted into a plasmid and expressed in a host).
What does the Reverse Transcriptase Assay contain?
MMLV RT Enzyme, RNase Inhibitor, and Poly-TTT Primer
What is the reference gene we used?