Transcriptional Analysis Flashcards
1
Q
Total RNA isolation
A
- Trizol
2. miRNeasy Mini Kit
2
Q
Total RNA composition
A
mRNA 5%
tRNA 15%
rRNA 80%
3
Q
RNA quality control
A
28S and 18S
RIN (RNA integrity number) score: 10
4
Q
RNAse Protection Assay
A
- Isolate RNA
- Hybridize with labeled probe targeting mRNA of interest
- Perform RNAse treatment to degrade single stranded RNA
- Gel Electrophoresis
- Detection
5
Q
Northern Blotting
A
- Isolate total RNA or mRNA
- Separate by size on agarose gel
- Transfer to membrane
- Hybridize using a labeled probe specific for gene of interest
- Detect
6
Q
Methods to analyze levels of specific mRNA
A
- RNAse Protection Assay
- Northern Blotting
- In Situ Hybridization
- RNA Scope
- PCR
7
Q
In Situ Hybridization
A
- Formalin fix cells or tissues of interest
- If tissue, make sections and apply to microscope slides
- Hybridize with labeled (fluorescent or biotinylated) probe
- Wash to remove unbound probe
- Detect using fluorescent microscope
8
Q
RNA Scope
A
- An advanced in situ hybridization technique
- Detects single RNA molecules and is quantitative at the single cell level
- Ideal for clinical applications
- Methodology very similar to ISH, but is amplifiable
9
Q
PCR
A
- Isolate RNA
- Generate cDNA
- Amplify cDNA using gene specific primers
- Run gel or quantitate qPCR signal
10
Q
cDNA synthesis
A
- Primer annealing
a. polyDT to bind polyA tail plus random primers - DNA polymerization
- Enzyme deactivation
11
Q
Standard PCR Amplification
A
- Denaturation - temperature increased to separate DNA strands
- Annealing - temperature decreased to allow primers to base pair to complementary DNA template
- Extension - Polymerase extends primer to form nascent DNA strand
