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Nutrition 400 > Using Tissue Culture and Molecular Techniques in Human Nutrition > Flashcards

Using Tissue Culture and Molecular Techniques in Human Nutrition Flashcards

1
Q

Types of nutrient-gene interactions (1 of 3) - Direct interactions

A

behave as transcription factors that can bind to DNA and induce gene expression

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2
Q

Types of nutrient-gene interactions (1 of 3) - Epigenetic interactions

A

alter the structure of DNA so that gene expression is altered

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3
Q

Types of nutrient-gene interactions (1 of 3)- genetic variations

A

common genetic variations such as single-nucleotide polymorphisms (SNPs) can alter the expression or functionality of genes

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4
Q

steps from sampling to PCR

A
  1. tissue sample (sampling)
  2. DNA, RNA ( nucleic acid isolation)
  3. cDNA (RT)
  4. PCR (real time PCR amplifications)
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5
Q

Reverse transcription

A

conversion of mRNA to cDNA - primer binds to mRNA and copies the first cDNA strand. Reverse transcription digests and displaces mRNA and copies second strand of DNA to end up with a double stranded cDNA.

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6
Q

Polymerase Chain Reaction (PCR)

A

amplifies specific, short DNA or mRNA sequences exponentially to allow detection and quantification of gene expression

  • denaturation, annealing, extension
  • each time increases by 2^1, 2^2, 2^3 etc for 30 cycles
  • plateau occurs when reagents are gone, polymerase is damaged or products accumulate
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7
Q

Types of Cell Culture (1 of 4) - Animal cell culture

A
  • animal cells taken from a living organisms and grown under controlled condition in vitro
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8
Q

Types of Cell Culture (1 of 4) - primary cell culture

A

cells isolated directly from the organisms

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9
Q

Types of Cell Culture (1 of 4) - cell line

A

derived from tumours or from cells transformed in vitro, although some of the very earliest lines were established from normal embryonic tissue

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10
Q

Types of Cell Culture (1 of 4) - immortal cell line

A

in vitro transformation

cells often lose the ability to differentiate

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11
Q

What is cell culture used for? (6)

A
  • model systems - cell biology study, interactions and effects of drugs, process of triggering aging
  • toxicity testing - effects of new drugs
  • cancer research
  • virology - cultivation of virus for vaccine
  • genetic engineering
  • gene therapy
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12
Q

Nutrients can…

A
  • influence stability of DNA
  • activate transcription factors
  • blind transporter protein s in the cytosol
  • influence signal transduction
  • alter gene expression and enzyme activities
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13
Q

Check results by gel electrophoresis

A
  • is product size what you expected?
  • is there more than 1 band?
  • Are there controls?
  • optimize the reaction conditions via - annealing temp of primers, concentration of Mg2+, extension time, amount of template and polymerase
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14
Q

Quantitative Real Time PCR and example

A
  • detect “how much” as they are generated in the reaction
  • requires a ssDNA probe with fluorescence - detection of probe occurs when strand is created and probe is removed from DNA - emits light
  • detection occurs quicker if original sample as more strands in it - from this curve, you can quantify how much of the DNA you started with in the sample
  • used to quantify the amount of virus in blood sample
    ex - detect how much CSF-1 expression occurs in tissues +/- DHA
  • SYBR green I assay and Taqman assay
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15
Q

Nutrigenomics

A

applying the science of molecular biology to human nutrition in order to understand the relationship between nutrition and health
- long term aim to understand how the whole body responds to nutrition using an integrated approach – systems biology *

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16
Q

Microassay

A
  • What are the differences in the genes expressed (unregulated or down regulated) in different samples
  • sample ⇒ DNA extraction ⇒ mRNA ⇒ RT ⇒cRNA labelled ⇒ array hybridization ⇒ image acquisition ⇒ data analysis
    -may take treated and untreated sample, add together to plate, cDNA then binds to complimentary sequence gene on the plate to identify which sample has which genes
    ex - investigate differences in gene expression in breast tissue with either omega 3 or omega 6
17
Q

Western Blot

A
  • measures the expression of a specific protein

ex - to detect if probiotics protect the epithelial barrier by maintaining tight junction proteins; test the expression of tight junction proteins with or without probiotics

18
Q

Western Blot process

A
  • proteins are separated first via gel electrophoresis
  • gel is placed on a membrane and transfer occurs
  • primary antibody detects thep protein of interest
  • secondary antibody with enzyme is added
19
Q

Western Blot - things to consider

A
  1. loading control?
  2. is the data quantified?
  3. How large are the changes reported?
  4. How much of the blot is shown?
20
Q

Immunohistochemistry

A
  • localization of protein in cells and tissues by using fluorophores
  • proteins ⇒ labelled primary antibody ⇒ fluorescent tag ⇒ light hit and is detected

ex - stain for detection and localization of liver modified proteins in ethanol induced liver failure
ex - +/- DHA detect for CD95 in lipid rafts of breast cancer cells

21
Q

ELISA - enyme-linked immunosorbent assay

A
  • based on specific recognition of the target/antigen compound by antibodies
  • detection of Ag-Ab complex with an enzyme labelled antibody or antigen
  • colour intensity of the enzyme colour reaction is proportional to the concentration of the analyte in the sample
    ex - DHA in trophoblasts - test for secretion of VEGF in the wells
    (DHA stimulates tube formation in the trophoblast cells)
22
Q

Types of ELISA

A

Direct - primary
Indirect - primary and secondary
Sandwich - 3 antibodies
Competitive - inhibitor antigen

23
Q

Flow Cytometry

A
  • laser based biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering
  • add specific binding to fluorescent antibodies to cell surface antigens of interest
  • pass though laser based tube in fluid - detector will sense different wavelengths immured from cells and count them
  • fresh garlic induces growth arrest/differentiation of breast cancer cells - label arrest cells, tube will then count them
24
Q

Advantages of flow cytometry

A
  • very large number of particles can be examined in a very short time
  • separates single particles physically from mixed populations
25
Q

Tansgenic animals

A

animal contains a foreign gene introduce purposely by human intervention
types ⇒ traditional transgenics
⇒ gene targetted - knock ins, knock outs

26
Q

transgenic animsl - examples

A

JCR:LA-cp rats - homogygous for the autosomal recessive cp gene, becomes obese and insulin resistant, with marked hyperinsulinemia and hyperTAG
ob/ob - spontaneous mutations on the ob gene
fat-1 mouse - for the study of n-3 PUFA - genetic mutation to convert n-6 to n-3 PUFA

27
Q

Summary - DNA

A

PCR

transgenic animals

28
Q

Summary - RNA

A
  • RT-PCR

- microarray / nutrigenomics

29
Q

Summary - protein

A
  • Western Blot
  • Immunohistochemistry
  • immunofluorescence
  • ELISA
  • FC
30
Q

Summary of examples

A

RT- PCR - detect how much (CSF-1 expression with +/- DHA, amount of viruses in blood etc)

western blot - measures expression of a protein (probiotics protect the epithelial barrier by maintaining tight junction proteins)

flow cytometry - cell counting (how many breast cancer cells are in the arrested state with garlic extract)

ELISA - detection of target antigens using antibodies (colour shows that it is there; +/- DHA trophoblasts secretion of growth factor VEGF)

immunohistochemistry - localization of proteins (ethanol induced liver failure - localization of modified proteins, or +/- DHA detect CD95 in lili rafts of breast cancer tissue -changed location)

microassay - differences in gene expression (breast cancer cells with omega 3 or omega 6 - up or down regulation of certain genes?)

Nutrition 400 (8 decks)

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