analysis of gene expression

Question 1

What is the first step in quantifying the level of expression of a specific gene?

Answer 1

Extract the RNA from the Cell

This is the preliminary step before any quantification methods can be applied.

Question 2

What is TRIzol used for?

Answer 2

Purification of Total Cellular RNA

TRIzol is a reagent used for RNA extraction.

Question 3

What is the purpose of the Qiagen Rneasy Mini Kit?

Answer 3

Column Purification of RNA

This kit is used to efficiently purify RNA from biological samples.

Question 4

What are the four steps involved in the column purification of RNA?

Answer 4

• Lyse
• Bind
• Wash
• Elute

These steps ensure effective isolation of RNA from contaminants.

Question 5

Which type of RNA is the largest amount found in a cell?

Answer 5

rRNA

In prokaryotes, the types include 23S, 16S, and 5S; in eukaryotes, 28S, 18S, 5.8S, and 5S.

Question 6

What does Northern blotting involve?

Answer 6

Size fractionation of RNA on a gel and detection using hybridisation of a specific probe.

This technique is used to study specific RNA molecules.

Question 7

What is the role of reverse transcriptase in RNA quantification?

Answer 7

It converts RNA into cDNA

This is essential for subsequent PCR amplification.

Question 8

What are the two major chemistries used in Quantitative (Real-Time) PCR (qPCR)?

Answer 8

• SYBR Green
• Taqman probes

SYBR Green binds to dsDNA, while Taqman probes fluoresce when degraded by Taq polymerase.

Question 9

How does qPCR differ from standard end-point PCR?

Answer 9

Quantification occurs at every cycle during amplification.

This allows for measurement of the starting template amount.

Question 10

What is the function of oligo dT primers in cDNA synthesis?

Answer 10

They bind to the 3’ polyA tail of mRNA.

This is specific to eukaryotic mRNA.

Question 11

What is the significance of using random primers in cDNA synthesis?

Answer 11

They bind at multiple places along all RNA sequences.

This allows for cDNA synthesis from various RNA types.

Question 12

What is the purpose of washing after hybridization in Northern blotting?

Answer 12

To remove non-specific interactions.

High stringency conditions are used to ensure specificity.

Question 13

What is the purpose of the microarray technique in RNA quantification?

Answer 13

To copy RNA into cDNA and hybridise to gene-specific DNA probes.

This allows for the analysis of gene expression profiles.

Question 14

What is RNA-seq used for?

Answer 14

Copying RNA into cDNA and sequencing cDNA fragments.

This technique provides insights into the transcriptome.

Question 15

What does the elution step in RNA purification involve?

Answer 15

Releasing RNA from the silica membrane.

This step collects the purified RNA for downstream applications.

Question 16

What is the significance of rRNA integrity in RNA preparation?

Answer 16

If rRNA is intact, it indicates a good quality preparation of total RNA.

Integrity is crucial for reliable downstream applications.

Question 17

What is the role of heat in cDNA synthesis?

Answer 17

It denatures the RNA in the presence of primers.

This prepares the RNA for reverse transcription.

Question 18

What type of primer is used to produce cDNA copies of a specific RNA sequence?

Answer 18

Gene-specific primer

This primer will only synthesize cDNA of the complementary RNA.

Question 19

What is the first step in quantifying the level of expression of a specific gene?

Answer 19

Make a cDNA copy of RNA using a DNA-dependent DNA polymerase.

This process is essential for converting RNA into a form that can be amplified and quantified.

Question 20

What are the two types of primers used in cDNA synthesis?

Answer 20

Random primers and oligo-dT primers.

Random primers bind at multiple places along all RNA sequences.

Question 21

What is the role of reverse transcriptase in cDNA synthesis?

Answer 21

It extends from the random primers joining complementary dNTPs together to produce cDNA fragments.

This process results in relatively short cDNA fragments of all RNA molecules.

Question 22

What are the two major chemistries used in Quantitative (Real-Time) PCR (qPCR)?

Answer 22

SYBR Green and Taqman probes.

SYBR Green binds to dsDNA, while Taqman probes fluoresce when degraded by Taq polymerase.

Question 23

What is the basic premise of qPCR?

Answer 23

The amplification of specific amplicons with quantification at every cycle.

This allows for measuring the amount of starting template during the exponential phase of amplification.

Question 24

How is relative quantification calculated in qPCR?

Answer 24

By calculating DCq, the difference in the number of PCR cycles to reach the threshold between the gene of interest and the reference gene.

The formula is DCq = (Cq_GOI - Cq_Reference).

Question 25

What does a larger Cq value indicate?

Answer 25

It indicates that more cycles are required to amplify the target to meet the threshold level, suggesting a lower amount of starting template. ## Footnote This is critical for understanding relative expression levels.

Question 26

What is the expression of hFIX relative to 16S rRNA in test cells with DCq of -2.76?

Answer 26

hFIX is expressed at nearly seven times the level of the reference gene. ## Footnote This is calculated using the formula 2^(-DCq).

Question 27

What does the term 'undetermined' mean in qPCR data?

Answer 27

It means the amplification has not crossed the threshold, indicating no target in the sample. ## Footnote Undetermined is not the same as zero.

Question 28

What is the significance of including a -RT control in qPCR?

Answer 28

It checks for contamination from genomic DNA, as amplification in the absence of reverse transcriptase indicates contamination. ## Footnote This is important for ensuring the specificity of the results.

Question 29

What does the formula 2^(-DDc) represent in the context of qPCR?

Answer 29

It represents the change in expression of a gene as a result of treatment. ## Footnote A negative DDc indicates an increase in expression.

Question 30

What does a primer efficiency (E) of 100% indicate?

Answer 30

It indicates that the primers are doubling the target DNA every cycle. ## Footnote This is calculated using E = 10^(-1/slope).

Question 31

What are microarrays used for?

Answer 31

They are used for gene expression analysis by hybridizing cDNA to specific DNA probes. ## Footnote Microarrays allow for simultaneous quantification of multiple genes.

Question 32

What is RNA-seq (WTSS)?

Answer 32

A method that involves isolating RNA from a cell sample, producing cDNA, and sequencing cDNA fragments. ## Footnote RNA-seq allows for comprehensive analysis of the transcriptome.

Question 33

What is the purpose of hybridizing samples with different fluorescent labels in microarrays?

Answer 33

To distinguish between cDNAs from different samples, allowing comparison of gene expression levels. ## Footnote This helps identify which genes are expressed differently in conditions such as disease.

Question 34

Fill in the blank: The difference in PCR efficiency (ΔE) of 3% between two targets generates a falsely calculated difference in the quantity ratio of ______ % after 25 cycles.

Answer 34

47

Question 35

True or False: The reference gene should have similar Cq values in control and test cells.

Answer 35

True

Question 36

What should be considered when designing primers for qPCR?

Answer 36

Primers should span an intron or flank an exon junction to ensure amplification from cDNA only. ## Footnote This prevents amplification from contaminating genomic DNA.

Question 37

What is the process of copying RNA into cDNA and hybridization to gene-specific DNA probes called?

Answer 37

Microarrays ## Footnote Microarrays are used for quantifying RNA in the transcriptome.

Question 38

What technique involves copying RNA into cDNA and sequencing of cDNA fragments?

Answer 38

RNA-seq ## Footnote RNA-seq is a method for quantifying levels of gene expression.

Question 39

What does WTSS stand for in RNA sequencing?

Answer 39

Whole Transcriptome Shotgun Sequencing ## Footnote WTSS is a specific type of RNA sequencing.

Question 40

What is the first step in the RNA-seq workflow?

Answer 40

Extract total RNA from cells ## Footnote This is crucial for obtaining the RNA sample needed for further analysis.

Question 41

What is an optional step in the RNA-seq workflow for eukaryotes?

Answer 41

Purify mRNA from total RNA ## Footnote Purifying mRNA can enhance the specificity of the analysis.

Question 42

What does Northern blotting involve?

Answer 42

Size fractionation of RNA on a gel and detection of RNA using hybridization of a specific probe ## Footnote Northern blotting is a method for quantifying target RNA of interest.

Question 43

What does QPCR stand for?

Answer 43

Quantitative Polymerase Chain Reaction ## Footnote QPCR is used for amplifying and quantifying RNA.

Question 44

Fill in the blank: The more a gene has been expressed in the cell sample, the greater the number of _______ molecules that would be present.

Answer 44

mRNA ## Footnote This indicates higher expression levels of the gene.

Question 45

What happens to cDNA fragments during RNA-seq?

Answer 45

They are sequenced using next-generation DNA sequencing chemistries ## Footnote This allows for the determination of gene expression levels.

Question 46

True or False: The greater the number of cDNA copies, the lower the expression level of the gene.

Answer 46

False ## Footnote More cDNA copies indicate a higher expression level of the corresponding gene.