Gene expression

Question 1

What is gene expression?

Answer 1

The process by which information encoded in a gene is converted into a functional product, usually RNA or protein.

Question 2

What is the central dogma of molecular biology?

Answer 2

DNA is transcribed into RNA, which is translated into protein.

Question 3

Why measure gene expression?

Answer 3

To compare healthy vs diseased tissue, tissue-specific expression, and effects of drugs or treatments.

Question 4

What is mRNA expression analysis used for?

Answer 4

To measure RNA levels, estimate genetic/environmental effects, and explain phenotype differences.

Question 5

Name three main methods to measure gene expression

Answer 5

qPCR, microarrays, and RNA sequencing.

Question 6

What scale is qPCR typically used for?

Answer 6

Small number of genes across many samples.

Question 7

Are qPCR values absolute or relative?

Answer 7

Relative values.

Question 8

Who invented PCR and when?

Answer 8

Kary Mullis in 1983.

Question 9

What enzyme is essential for PCR?

Answer 9

Heat-stable DNA polymerase (Taq polymerase).

Question 10

What are the three main PCR steps?

Answer 10

Denaturation, annealing, and extension.

Question 11

What temperature is used for PCR denaturation?

Answer 11

Approximately 90°C.

Question 12

What temperature is used for PCR annealing?

Answer 12

Approximately 54°C.

Question 13

What temperature is used for PCR extension?

Answer 13

Approximately 72°C.

Question 14

Why is PCR considered exponential?

Answer 14

Each cycle doubles the amount of target DNA.

Question 15

What is a good primer?

Answer 15

One that is specific, has suitable Tm, no dimers or hairpins, and minimal secondary structure.

Question 16

What is primer uniqueness?

Answer 16

The primer binds to only one target site in the genome.

Question 17

Typical primer length?

Answer 17

17–28 bases.

Question 18

Ideal GC content for primers?

Answer 18

Approximately 50–60%.

Question 19

What is melting temperature (Tm)?

Answer 19

The temperature at which half of DNA strands are single-stranded.

Question 20

How does GC content affect Tm?

Answer 20

Higher GC content increases Tm.

Question 21

Formula for annealing temperature?

Answer 21

Tanneal = Tm − 4°C.

Question 22

Why avoid primer dimers and hairpins?

Answer 22

They reduce PCR efficiency.

Question 23

How closely should primer pair annealing temperatures match?

Answer 23

Within 2–3°C.

Question 24

What is quantitative RT-PCR?

Answer 24

PCR that measures RNA expression levels in real time.

Question 25

What is a housekeeping gene?

Answer 25

A gene with stable expression used as a reference.

Question 26

Give examples of housekeeping genes

Answer 26

ACTB, GAPDH, 18S rRNA, UBC.

Question 27

What is Ct (cycle threshold)?

Answer 27

The cycle number at which fluorescence crosses the detection threshold.

Question 28

What are technical replicates?

Answer 28

Repeated measurements of the same sample.

Question 29

What are biological replicates?

Answer 29

Independent samples from different biological sources.

Question 30

What is ΔCt?

Answer 30

Ct(gene of interest) − Ct(housekeeping gene).

Question 31

What is ΔΔCt?

Answer 31

ΔCt(sample) − mean ΔCt(control).

Question 32

What does 2^(-ΔΔCt) represent?

Answer 32

Relative fold change in gene expression.

Question 33

What does a 2^(-ΔΔCt) value of ~2 indicate?

Answer 33

Two-fold upregulation.

Question 34

When are ΔCt values considered reliable?

Answer 34

When within 0.3 of the group median.

Question 35

What should be done if qPCR data are unreliable?

Answer 35

Use geometric mean, remove outliers, or repeat the experiment.

Question 36

What is a microarray?

Answer 36

A solid surface containing DNA probes to measure gene expression.

Question 37

What does microarray intensity represent?

Answer 37

Relative abundance of target sequences.

Question 38

Difference between one-channel and two-channel arrays?

Answer 38

One-channel measures single sample intensity; two-channel compares two samples.

Question 39

What dyes are used in two-channel microarrays?

Answer 39

Cy3 and Cy5.

Question 40

What is hybridization?

Answer 40

Binding of complementary nucleic acid sequences.

Question 41

What is comparative hybridization?

Answer 41

Direct comparison of two samples on the same array.

Question 42

Name common microarray issues

Answer 42

Scratches, air bubbles, spatial bias, background noise.

Question 43

What is background correction?

Answer 43

Adjustment for non-specific hybridization.

Question 44

What is spatial bias?

Answer 44

Intensity variation due to probe location on the array.

Question 45

What is normalization?

Answer 45

Adjusting data to allow comparison between samples.

Question 46

What is quantile normalization?

Answer 46

Method that aligns distributions across samples.

Question 47

Why use log2 ratios in microarrays?

Answer 47

To reduce dye bias and normalize distributions.

Question 48

What is dye bias?

Answer 48

Systematic intensity differences between Cy3 and Cy5 dyes.

Question 49

What is Bioconductor?

Answer 49

An R project for bioinformatics data analysis.

Question 50

What is the goal of gene expression profiling?

Answer 50

Identify differentially expressed genes.

Question 51

What statistical tests are used for expression analysis?

Answer 51

t-tests, ANOVA, RankProducts.

Question 52

What is a Type I error?

Answer 52

False positive (detecting change by chance).

Question 53

How is Type I error reduced?

Answer 53

Bonferroni correction.

Question 54

What is a Type II error?

Answer 54

False negative (missing true change).

Question 55

How is Type II error reduced?

Answer 55

Benjamini–Hochberg FDR correction.

Question 56

What is RNA sequencing?

Answer 56

A high-throughput method to sequence and quantify RNA.

Question 57

Why is RNA-seq considered more powerful?

Answer 57

It detects novel and spliced transcripts and all RNA types.