W11 Nucleic Acid Biochemistry and RNA Interference Flashcards
isolation and purification of dna
homogenise cells/tissues in 4 degrees with sterile equipment
cellular lysis using detergent or lysozyme
chelating agents like EDTA or citrate to inhibit DNases
proteinase agents like proteinase K for enzymatic digestion of proteins
phenol extraction to remove proteins
alcohol precipitation with 70%/100% ethanol or isopropanol
redissolve DNA in TE buffer
isolation and purification of rna
treat reagents with RNase inhibitors
homogenise cells/tissues in 4 degrees
cellular lysis using detergent or lysozyme
rna solvents and proteinase agents
phenol extraction
alcohol precipitation
redissolve rna
what is fluorometry
method why dyes that bind specifically to dna/rna or proteins undergo chemical change > emit light when exposed to certain wavelength of light > amount of fluorescence proportional to concentration of target molecule
dyes that are used in fluorometry
SYBR green: bind to ds dna
SYBR gold: bind to both rna and dna
hoechst 33258: bind to ds dna
picogreen: bind to ds dna
ribogreen rna reagent: bind to rna only
what is RNA interference
process by which expression of a target gene is effectively silenced or knocked down by the selective inactivation of its corresponding mRNA by double stranded rna
requirements for RNAi
the inducer: dsRNA
target mRNA degradation is sequence-specific and homology dependent
key players in machinery:
- Dicer (ribonuclease, RNase III enzyme) to process dsRNA into short RNAs
- Drosha (RNase III) used to process shRNA into siRNA
- rna-induced silencing complex (RISC, ribonucleoprotein) to bind to RNA of interest to degrade it
properties of using siRNAs for gene silencing
rapid and transient knockdown (3-7 days) of gene expression
applicable in cell lines that are amenable to transfection by liposomes/electroporation
amount of siRNA introduced is highly controlled
efficiency of gene knockdown is dependent on transfection efficiency, siRNA stability and half-life of protein target
how are shRNA used for RNAi
contains a loop structure that is processed to siRNA
vector-based, rna transcribed from dna vectors
exogenously introduced into cells by viral transduction
degradation of mRNAs upon complementary binding of target mRNA
sequence specific gene silencing
properties of using shRNAs for gene silencing
prolonged knockdown of gene expression
used in cell lines difficult to transfect
introduction by viral vectors allows for stable integration of shRNA expression vectors through use of antibiotic selection markers
control of shRNA expression using inducible promoters
ability to co-express shRNAs with a reporter gene
applications of RNAi
gene knockdown
functional genomics
transgenic plants
cancer
neurological diseases
viral infection
