Transcriptional Analysis

Question 1

Total RNA isolation

Answer 1

1. Trizol

2. miRNeasy Mini Kit

Question 2

Total RNA composition

Answer 2

mRNA 5%
tRNA 15%
rRNA 80%

Question 3

RNA quality control

Answer 3

28S and 18S

RIN (RNA integrity number) score: 10

Question 4

RNAse Protection Assay

Answer 4

1. Isolate RNA
2. Hybridize with labeled probe targeting mRNA of interest
3. Perform RNAse treatment to degrade single stranded RNA
4. Gel Electrophoresis
5. Detection

Question 5

Northern Blotting

Answer 5

1. Isolate total RNA or mRNA
2. Separate by size on agarose gel
3. Transfer to membrane
4. Hybridize using a labeled probe specific for gene of interest
5. Detect

Question 6

Methods to analyze levels of specific mRNA

Answer 6

1. RNAse Protection Assay
2. Northern Blotting
3. In Situ Hybridization
4. RNA Scope
5. PCR

Question 7

In Situ Hybridization

Answer 7

1. Formalin fix cells or tissues of interest
2. If tissue, make sections and apply to microscope slides
3. Hybridize with labeled (fluorescent or biotinylated) probe
4. Wash to remove unbound probe
5. Detect using fluorescent microscope

Question 8

RNA Scope

Answer 8

1. An advanced in situ hybridization technique
2. Detects single RNA molecules and is quantitative at the single cell level
3. Ideal for clinical applications
4. Methodology very similar to ISH, but is amplifiable

Question 9

PCR

Answer 9

1. Isolate RNA
2. Generate cDNA
3. Amplify cDNA using gene specific primers
4. Run gel or quantitate qPCR signal

Question 10

cDNA synthesis

Answer 10

1. Primer annealing
   a. polyDT to bind polyA tail plus random primers
2. DNA polymerization
3. Enzyme deactivation

Question 11

Standard PCR Amplification

Answer 11

1. Denaturation - temperature increased to separate DNA strands
2. Annealing - temperature decreased to allow primers to base pair to complementary DNA template
3. Extension - Polymerase extends primer to form nascent DNA strand