BCH5413 EXAM 3 ACTUAL ASSESSMENT TEST
2026 TESTED QUESTIONS WITH FULL
SOLUTION GRADED A+
◉ delta-delta Ct method. Answer: [delta-Ct(treated sample)] -
[delta-Ct(control sample)]
◉ delta-Ct. Answer: Ct(gene) - Ct(endogenous control)
◉ RT-PCR: semi-quantitative v. quantitative. Answer: Semi-
quantitative
- "regular"
Quantitative
- graphing the RT-PCR
- is a form of analysis of data
◉ Measuring changes in gene expression
- using RT-PCR v. Northern blot. Answer: Northern blot
- in example, took up to 2 months
RT-PCR
,- in example, took up to 2 days
◉ Microarray analysis
- allows you to.... Answer: measure amounts of a nucleic acid species,
not one at a time on a Northern blot or RT-PCR, but every expressed
gene in a cell at one time
◉ Two different microarray's. Answer: cDNA and oligonucleotide
◉ Microarrays: RNA isolation
- RNA must be.... Answer: - RNA must be high quality, free of
DNA/contamination (A260/280 ratio; should be above 1.5)
◉ Microarrays: RNA isolation
- techniques. Answer: Clontech column-based
- start with sample
- homogenize, centrifuge, filter
- wash column and collect purified RNA
Invitrogen TRizol reagent
- homogenize sample in TRizole
- phase separation
,- RNA precipitation
- wash and solubilize RNA
- degrade any genomic DNA that came through purification steps
◉ cDNA Microarrays
- steps. Answer: - glass slide arrays are usually probed with
fluorescent cDNA
- by using two different color dyes (red and green), each RNA serves
as its own control
- if the spot is red, one probe is more abundant
- if the spot is green, the other probe is more abundant
- if the spot is yellow, then the two probes are equally abundant
Microarrays:
◉ cDNA Microarrays
- colored dyes and steps. Answer: - cDNA reverse transcribed from
mRNA and labeled with a fluorescent dye
- the control sample without serum has green dye
- treated sample with serum has red dye
- both were mixed and incubated with microarray
- microarray washed
- fluorescence measured on chip
, ◉ cDNA Microarrays
- if a spot is green, expression is...
- if a spot is red, expression is.... Answer: - if a spot is green,
expression of that gene decreases in cells after serum addition
- if a spot is red, expression of that gene increases in cells after
serum addition
◉ cDNA Microarrays
- what each colored dye means. Answer: - Red = gene expression
increased
- Green = gene expression decreased
- Yellow = no change (expression equal)
- Blue/Black = low to no expression
◉ Oligonucleotide Microarrays: Affymetrix GeneChip
- what is it/how it works. Answer: - short oligonucleotides are
synthesized on a silicon chip, using the same kind of
photolithography process used to make microprocessors
- About 15-20 match and mismatch oligos are made for each gene to
be probed; each oligo is 20nt in length
◉ Labeling your target for GeneChip/oligonucleotide analysis
2026 TESTED QUESTIONS WITH FULL
SOLUTION GRADED A+
◉ delta-delta Ct method. Answer: [delta-Ct(treated sample)] -
[delta-Ct(control sample)]
◉ delta-Ct. Answer: Ct(gene) - Ct(endogenous control)
◉ RT-PCR: semi-quantitative v. quantitative. Answer: Semi-
quantitative
- "regular"
Quantitative
- graphing the RT-PCR
- is a form of analysis of data
◉ Measuring changes in gene expression
- using RT-PCR v. Northern blot. Answer: Northern blot
- in example, took up to 2 months
RT-PCR
,- in example, took up to 2 days
◉ Microarray analysis
- allows you to.... Answer: measure amounts of a nucleic acid species,
not one at a time on a Northern blot or RT-PCR, but every expressed
gene in a cell at one time
◉ Two different microarray's. Answer: cDNA and oligonucleotide
◉ Microarrays: RNA isolation
- RNA must be.... Answer: - RNA must be high quality, free of
DNA/contamination (A260/280 ratio; should be above 1.5)
◉ Microarrays: RNA isolation
- techniques. Answer: Clontech column-based
- start with sample
- homogenize, centrifuge, filter
- wash column and collect purified RNA
Invitrogen TRizol reagent
- homogenize sample in TRizole
- phase separation
,- RNA precipitation
- wash and solubilize RNA
- degrade any genomic DNA that came through purification steps
◉ cDNA Microarrays
- steps. Answer: - glass slide arrays are usually probed with
fluorescent cDNA
- by using two different color dyes (red and green), each RNA serves
as its own control
- if the spot is red, one probe is more abundant
- if the spot is green, the other probe is more abundant
- if the spot is yellow, then the two probes are equally abundant
Microarrays:
◉ cDNA Microarrays
- colored dyes and steps. Answer: - cDNA reverse transcribed from
mRNA and labeled with a fluorescent dye
- the control sample without serum has green dye
- treated sample with serum has red dye
- both were mixed and incubated with microarray
- microarray washed
- fluorescence measured on chip
, ◉ cDNA Microarrays
- if a spot is green, expression is...
- if a spot is red, expression is.... Answer: - if a spot is green,
expression of that gene decreases in cells after serum addition
- if a spot is red, expression of that gene increases in cells after
serum addition
◉ cDNA Microarrays
- what each colored dye means. Answer: - Red = gene expression
increased
- Green = gene expression decreased
- Yellow = no change (expression equal)
- Blue/Black = low to no expression
◉ Oligonucleotide Microarrays: Affymetrix GeneChip
- what is it/how it works. Answer: - short oligonucleotides are
synthesized on a silicon chip, using the same kind of
photolithography process used to make microprocessors
- About 15-20 match and mismatch oligos are made for each gene to
be probed; each oligo is 20nt in length
◉ Labeling your target for GeneChip/oligonucleotide analysis
