Determination of mRNA Expression

Question 0

Levels where gene expression is regulated

Answer 0

Chromatin remodeling
Transcription
RNA procession
mRNA stability
Post-translational modification

Question 1

Why study mRNA expression

Answer 1

Cell functions governed by gene expression
Basis of cell diff, morph and adaptability
NB in disease pathogenesis
Diagnostic/ prognostic value

Question 2

What modifies gene expression

Answer 2

Heritable factors
Cellular cues eg. TF, signaling pathways
Internal factors eg. Tumour
External factors eg. Air pollution

Question 3

Why RNA more prone to hydrolysis

Answer 3

Extra hydroxyl group

Question 4

Types of RNa

Answer 4

Ribosomal
Transfer
Messenger
Micro (antisense RNA that regulate expression)
Long non coding (also regs of gene expression)

Question 5

How does miRNA work

Answer 5

Mature miRNA couples with RISC (RNA induced silencing complex) and degrades RNA by cleavage / inhibits protein synthesis

Question 6

Egs of long non coding RNA

Answer 6

XIST RNA - inhibits X chromosome

HOX antisense intergenic RNA (HOTAIR) inhibits transcription through Epigenetic regulation

Question 7

Why critical to have separate RNA area in lab

Answer 7

RNA is unstable

RNAses common contaminant

Question 8

How to protect against RNases

Answer 8

Gloves
RNA free equipment and chemicals
Sterile technique 
Treat everything with DEPC
Heat treatment of materials
Use RNA inhibitors in reactions

Question 9

What is DEPC

Answer 9

Diethylpyrocarbonate
Irreversibly interacts with histidine in RNAse active site.
Very toxic
Can inhibit downstream apps eg. qRT-PCR

Question 10

Steps in RNA extraction

Answer 10

1. Sample collection (freeze or use fixative)
2. Tissue disruption and cell lysis (lysis buffers/ manual homogenizing)
3. Release and protection of RNA
4. Purification and concentration of RNA
5. Storage of RNA (on ice/ precipitated in ethanol)

Question 11

Ways to protect released RNA

Answer 11

Guanidium Thiocyanate (protein denaturant, also help bind to silica)
B-mercaptoethanol/ SDS
DNase 1 
Phenol chloroform extraction
Isopropanol precipitates nucleic acids

Question 12

RNA concentration (NanoDrop)=

Answer 12

OD x DF x 40ug/mL

Question 13

How do you verify RNA quality

Answer 13

Formaldehyde gel electrophoresis (two clear bands = intact 28S and 18S)
Bioanalyser (uses on chip capillary electrophoresis, less sample needed)

Question 14

Ways to analyze mRNA

Answer 14

Northern blot
Semi qRT-PCR
qRT-PCR
Microarray

Question 15

How NB works

Answer 15

Run RNA gel
Transfer to nitrocellulose membrane by capillary action
Label with radioactive probe
Image

Question 16

Disadv of NB

Answer 16

Hazardous chemicals
Long time
RNA may degrade

Question 17

What is a CT value

Answer 17

Crossing threshold value

Point at which fluorescence enters log phase of amplification in qRT-PCR

Question 18

What is the diff between relative quantification and absolute quantification

Answer 18

Relative compares the change in CT between a housekeeping gene and the target gene. This is done for all samples.

Absolute uses a standard curve of log fold dilutions to compare CT value of unknown sample.

Question 19

Adv and disadvantage of in situ hydrydization

Answer 19

Adv - detect mRNA expression in cell/ tissue

Disadv - difficult to quantitate levels