b. Tutorial 2: RNA modifications and Quantifying RNA

Question 1

Describe gene expression

Answer 1

process in which the info encoded in a gene is converted into a phenotype

Question 2

Describe transcription

Answer 2

the process of producing RNA from DNA

Question 3

Describe translation

Answer 3

the process of producing a protein from RNA

Question 4

They say that all the cells in an indiv contains the same DNA so how can cells vary?

Answer 4

While they all contain the same genes not all of those genes are turned on. Thus allowing for variation

Question 5

“some genes are ubiquitously expressed” what does this mean?

Answer 5

this phrase is referring to the genes that are expressed (turned on) in all cell types

Question 6

What causes certain genes to be expressed vs not expressed in certain cells?

Answer 6

the cellular condition of that cell type

Question 7

How is gene expression of a cell monitored?

Answer 7

using the level of mRNA present

Question 8

Describe a Gene expression profile

Answer 8

identifying the mRNA levels present for many genes simultaneously

Question 9

How is cDNA produced?

Answer 9

By taking an RNA strand and using the reverse transcriptase PCR to convert that unstable RNA strand to stable cDNA so it can be amplfied

Question 10

What does RT-PCR stand for?

Answer 10

reverse transcriptase Polymerase chain rxn

Question 11

Describe qPCR

Answer 11

PCR but you add fluorescent dye to visualize the amplification processes.

Question 12

Describe CT values. Which type of PCR is this associated w/?

Answer 12

a) The number of PCR cycles required for the fluorescence to be reach threshold. Which is used to quantify the amount of initial [DNA]
b) quantitative PCR

Question 13

T or F - The CT values is directly proportional to the amount of template DNA in the initial sample

Answer 13

F - it is inversely proportional

Question 14

What two things does qRT-PCR combine?

Answer 14

1. qPCR = using fluorescence to detect initial template DNA amounts
2. RT-PCR = converting RNA to cDNA using Reverse Transcriptase

Question 15

What is the purpose of qRT-PCR?

Answer 15

its a techniques that’s used to analyze mRNA levels of a specific gene

Question 16

How does one use qRT-PCR to measure the amount of gene expression of a target gene in 5 steps?

Answer 16

1. After gene is transcribed isolate all of the mRNA produced
2. Use the reverse transcriptase to convert the mRNA to cDNA
3. anneal forward and reverse primers on to the cDNA after its denatured
4. Use the qRCR to amplify the cDNA until the fluorescent signal reaches threshold
5. Calculate amount of gene expression by using the inverse of the CT value (number of cycles needed to reach threshold)

Question 17

T or F - there is no limit to using qRT-PCR to quantify gene expression

Answer 17

F - it can only analyze one gene at a time

Question 18

You are testing the efficacy of a new drug treatment to block the expression of a particular problematic gene. You test cells with different doses of this treatment, collect total RNA from each treatment group and use qRT-PCR to quantify the results, below. Which treatment dose is the most effective at blocking expression of this gene?

Answer 18

50 mg (brown). This amplification curve passes above the threshold of detection at about cycle 35, indicating a Ct value of 35. Remember that a high Ct value indicates relatively low levels of the initial mRNA in the sample. Therefore, the treatment must have DEC expression as a DEC amount of mRNA was originally produced.

Question 19

You are using are testing the efficacy of a new drug treatment to block the expression of a particular problematic gene. You test cells with different doses of this treatment, collect total RNA from each treatment group and use qRT-PCR to quantify the results, below. What is the Ct value of the 20 mg treatment does?

Answer 19

23. This amplification curve (green) passes above the threshold of detection at about cycle 23, indicating a Ct value of 23.

Question 20

What is the purpose of RNA-Sequencing (RNA-seq)?

Answer 20

a technique used to quantify and compare genome wide gene expression

Question 21

How does RNA-Seq quantify and compare genome wide gene expression in 5 steps?

Answer 21

1. After transcription the RNA is isolated from sample
2. The RNA is converted to cDNA via RT-PCR
3. the cDNA is sequenced via next generation sequencing (where they anneal the fragment to a bead, place it in a well w/ a bunch of dNTPs and computer reads the flashes of light that shine when is synthesized due to the luciferase reacting w/ the luciferin in the well sol’n when ATP is present)
4. a computer aligns the generated sequences (reads) to a reference genome to determine the gene that that RNA was transcribed from
5. the number of sequences that were generated from each RNA fragment that aligned w/ a particular gene indicates the level of expression for that gene

Question 22

Examine the RNA-seq data below. Here the viral reads are plotted on the y-axis and a schematic of the viral genome is plotted along the x-axis. Which treatment day has the lowest viral load (level of viral RNA present)?

Answer 22

Day 1 has very low levels of viral reads, with some regions of the genome having no reads. This indicates that some regions of the viral genome are not expressed at all at this time point.

Question 23

Which tissue has the highest expression of the gene under examination in the figure below?

Answer 23

Kidney. We can see that this tissue had the highest number of reads at about 35 RPKM.