DNA, RNA and protein isolation

Question 1

different disease modelling approaches (4)

Answer 1

• in silico (computational approaches)
• in vitro (controlled environment)
• ex vivo
• in vivo (animal models)

Question 2

what are some examples of in vitro experimentation?

Answer 2

• cells isolated from multicellular organisms
• subcellular components (e.g. mitochondria, ribosomes)
• purified moleules (DNA, RNA or proteins)

Question 3

two examples of cells in cultures

Answer 3

• HEK293T; human embryonic kidney origin, continuous cell line
• LEC; human dermal lymphatic endothelial cells; primary cells

Question 4

what are primary cultures?

Answer 4

• isolated directly from human/animal tissue
• finite lifespan
• some cells e.g. tumor, have capacity to grow indefinitely in culture

Question 5

what are cell lines?

Answer 5

• continuously passaged over a long period of time
• cells can be immortalised by specific culture conditions, or addition of other genes (often oncogenes)
• lots of cell lines exist, derived from various tissues
  -usually possess many/some characteristics of the original tissue, but may not be entirely representative

Question 6

factors in selecting the appropriate cell line.

Answer 6

• species specific cultures
• functional characteristics; liver and kidney suitable for toxicity testing
• finite or continuous
• growth conditions and characteristics

Question 7

what can finite cell lines do?

Answer 7

maintain tissue like appearance

Question 8

what is a benefit of continuous cell lines?

Answer 8

easier to clone and maintain

Question 9

what can cell cultures with a fast growth rate be used for?

Answer 9

for the expression of a recombinant protein in high yields

Question 10

what are the applications of cell cultures?

Answer 10

• normal physiology and biochemistry of cells e.g. metabolic studies
• effects of drug and toxic compounds of cells (drug screening and development)
• mutagenesis
• large scale manufacturing of biological compounds e.g. vaccines and therapeutic proteins

Question 11

what are three limitations of 2D cell monolayers in growing cell cultures?

Answer 11

• cell lose their phenotype
• lack cell-cell and cell-matrix interactions
• cant mimic cellular functions and signalling pathways like in vivo conditions

Question 12

what are organoids?

Answer 12

3D cell aggregates derived from primary tissue or stem cells

Question 13

what are spheroids?

Answer 13

often formed from cancer cell lines or tumour biopsies

Question 14

what are 3 advantages of 3D cultures in growing cells in cultures?

Answer 14

• more similar to in vivo conditions
• more realistic biochemical and physiological responses
• tumor organoids shown to predict how well patients response to cancer drugs to aid in personalised medicine

Question 15

what is a biosafety cabinet used for? (fume hood)

Answer 15

• personal protection from harmful agents within the cabinet
• product protection to avoid contamination of the samples
• everything inside has to be sprayed with 70% EtOH

Question 15

what are two prinicples used in cell culture?

Answer 15

• maintain aseptic conditions
• sterile handling and storage of cell culture, reagents and media

Question 16

what is the physiological environement used in cell culture?

Answer 16

• growth medium ; provides nutrients (aa, carbs, vitamins, minerals), GFs, hormones to regulate pH/osmotic pressure

Question 17

what is the physiochemical environment in a cell culture that is maintained?

Answer 17

• pH; 7.4 for most mammalian cell cultures
• CO2; 5-7% for most cell cultures
• temp; 37 celsius

Question 18

what do fibroblastic-like cells look like under light microscopy?

Answer 18

• elongated shape
• grow attached to the substrate

Question 19

what do epithelial-like cells look like under light microscopy?

Answer 19

• polygonal in shape
• regular dimensions
• grow attached to a substrate in discrete patches

Question 20

what do lymphoblast-like cells look like under light microscopy?

Answer 20

• spherical in shape
• grown in suspenstion (no attachment to surface)

Question 21

what is passaging cell cultures?

Answer 21

the process of harvesting cells from a culture, transferring the cells to one of more culture vessels with fresh growth medium and using those cells to start new cultures

Question 22

how do we isolate DNA, RNA and proteins from the cell cultures?

Answer 22

two protocols; solution-based and column-based
- commercially available complete kits. most require repeated centrifugatio steps followed by removal of supernatants depending on type of speciment and extra mechanical treatment

Question 23

isolation phases in solution based separation

Answer 23

aqueous phase; RNA
interphase; DNA
organic phase; proteases and lipids

separate depending on weight, lightest at the top

Question 24

what do we use to measure DNA, RNA and protein concentration and quallity?

Answer 24

spectrophotometric analysis

Question 25

what is spectrophotometric analysis based on?

Answer 25

the principles that nucleic acids absorb UV light in a specific pattern.

Question 26

what happens in spectrophotometric analysis in case of DNA and RNA?

Answer 26

sample is exposed to UV light at wavelength of 260nm and photo-detector measures the light that passes through the sample.
- some will pass through and some absorbed, the more light absorbed by sample, the higher the nucleic acid conc in the sample.
= less light will hit the photodetector and will produce a higher optical density (OD).

Question 27

what does absorbance at 260nm mean?

Answer 27

nucleic acids absorb at 260nm due to aromatic base moieties within their structure, purines (thymine, cytosine and uracil) and pyrimidines (adenine and guanine)

Question 28

what does absorbance at 280nm mean?

Answer 28

typically where proteins and phenolic compounds have strong absorbance. Aromatic amino acid side chains (tryptophan, phenylalanine, tyrosine and histidine) within proteins are responsible for this absorbance

Question 29

what does absorbance at 230nm mean?

Answer 29

many organic compounds have strong absorbances at around 225nm.
in addition to phenol and TRIzol, and chaotropic salts, the peptide bonds in protiens absorb light between 200-230nm

Question 30

what is the A260/280 ratio used for?

Answer 30

to determine protein contamination of a nucleic acid sample.
for pure RNA and DNA this ratio should be somewhere around 2.0 and 1.8, respectively.
Lower means sample is protein contaminated.

Question 31

what does a A260/230 ratio indicate?

Answer 31

the presence of organic contaminants such as phenol, TRIzol, chaotropic salts and other aromatic compounds.
Samples with this ratio below 1.8 are considered to have significant amount of these contaminants that will interfere with downstream applications,
In a pure sample this ratio should be close to 2.0

Question 32

what is a bradford protein assay?

Answer 32

quick and accurate spectroscopic analytical procedure used to measure conc of protein in a solution.
Reaction is dependent on the aa composition of the measured proteins.

Question 33

process of a bradford protein assay

Answer 33

colorimetic protein assay based on the dye Coomassie Brilliant Blue G250.
under acidic conditions, the red form of the dye is converted into its blue form after binding to the assayed protein

Question 34

summary of DNA, RNA and protein isolation

Answer 34

• cells in culture widely used in research as in vitro models since they can be isolated from animals/humans and grown in culture with supplied nutrients and GFs
• can differentiate between primary or immortalised cell lines that can be cultured in 2D or 3D
• selecting appropriate cell line depends on several factors and specially on final application of the cell culture
• cells in culture require sterile handling and specific environmental conditions
• we can isolate DNA, RNA and proteins from several biological samples using solution or column based methods
• quality and integrity of the sample affects results of downstream applications