Biological Techniques
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Where are cell lines derived from?
Usually tumours
What type of cell line is HeLa cells?
Cervical carcinoma from an aggresive tumour, transformed by human papilloma virus 18
Do HeLa cells have a regular number of chromosomes?
No
What are induced pluripotent stem cells
Reprogrammed adult somatic cells
How are iPS made?
Fibroblasts are isolated and transduced with transcription factors
Why transfect cells with nucleic acids
Protein of interest may not be expressed in cells to be studied, may not be an antibody to detect protein, study mutant
How is RNA interference (RNAi) to knockdown gene expression
Uses small interfering RNAs (siRNAs) to knockdown mammalian gene expression. Antisense siRNA strand is bound bt RNA-induced silencing complex (RISC) which cleaves mRNAs
Methods of transfection by mammalian cells
Electroporation Lipid based reagents Calcium phosphate –> forms complex w/ dna Injection of nucleic acids
What are electron microscopy sections stained with
Antibodies labelled with gold
Cryo-electron tomography has what resolution?
Near atomic
What does NGS use to generate short sequence reads?
Massive parallel sequencing
What does transcriptomics study?
mRNA molecules inside organisms, tissues, cells
How is transcriptisome measured?
cDNA microarrays
What do cDNA microarrays consist of>
convert mRNA to cDNA from 2 samples, label, spot on slide with cDNA probes.
How RNA-Seq works
Analyses cDNA copies from RNA by DNA sequencing methods, then reads are mapped onto reference genome.
SILAC meaning and what it analyses
stable isotope labeling amino acids in cell culture, proteome
What amino acids does SILAC tag?
Lysine and arginine