Using Tissue Culture and Molecular Techniques in Human Nutrition Flashcards
Types of nutrient-gene interactions (1 of 3) - Direct interactions
behave as transcription factors that can bind to DNA and induce gene expression
Types of nutrient-gene interactions (1 of 3) - Epigenetic interactions
alter the structure of DNA so that gene expression is altered
Types of nutrient-gene interactions (1 of 3)- genetic variations
common genetic variations such as single-nucleotide polymorphisms (SNPs) can alter the expression or functionality of genes
steps from sampling to PCR
- tissue sample (sampling)
- DNA, RNA ( nucleic acid isolation)
- cDNA (RT)
- PCR (real time PCR amplifications)
Reverse transcription
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.
Polymerase Chain Reaction (PCR)
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
Types of Cell Culture (1 of 4) - Animal cell culture
- animal cells taken from a living organisms and grown under controlled condition in vitro
Types of Cell Culture (1 of 4) - primary cell culture
cells isolated directly from the organisms
Types of Cell Culture (1 of 4) - cell line
derived from tumours or from cells transformed in vitro, although some of the very earliest lines were established from normal embryonic tissue
Types of Cell Culture (1 of 4) - immortal cell line
in vitro transformation
cells often lose the ability to differentiate
What is cell culture used for? (6)
- 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
Nutrients can…
- influence stability of DNA
- activate transcription factors
- blind transporter protein s in the cytosol
- influence signal transduction
- alter gene expression and enzyme activities
Check results by gel electrophoresis
- 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
Quantitative Real Time PCR and example
- 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
Nutrigenomics
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 *
Microassay
- 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
Western Blot
- 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
Western Blot process
- 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
Western Blot - things to consider
- loading control?
- is the data quantified?
- How large are the changes reported?
- How much of the blot is shown?
Immunohistochemistry
- 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
ELISA - enyme-linked immunosorbent assay
- 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)
Types of ELISA
Direct - primary
Indirect - primary and secondary
Sandwich - 3 antibodies
Competitive - inhibitor antigen
Flow Cytometry
- 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
Advantages of flow cytometry
- very large number of particles can be examined in a very short time
- separates single particles physically from mixed populations
Tansgenic animals
animal contains a foreign gene introduce purposely by human intervention
types ⇒ traditional transgenics
⇒ gene targetted - knock ins, knock outs
transgenic animsl - examples
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
Summary - DNA
PCR
transgenic animals
Summary - RNA
- RT-PCR
- microarray / nutrigenomics
Summary - protein
- Western Blot
- Immunohistochemistry
- immunofluorescence
- ELISA
- FC
Summary of examples
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?)
