methods to study gene expression

Question 1

measure rna level

Answer 1

for one or few - northern blot
-RT-PCR and qPCR

all rnas - micro arrays / rna sequencing

Question 2

northern blot

Answer 2

rna loaded into gel
gel run through electric field to seperate rna by size
filter placed on gel then weight on top to transfer rna to filter

add probe to rna and run through auto radiograph study results

Question 3

RT-PCR

Answer 3

DNA polymerases need DNA as template
RNA converted to DNA using reverse transcriptase
needs primers
random primer
oligo primer
sequence specific primer

then pcr
denaturing step - double helix denatured into 2 ss
annealing step - primer binds to each ss
extension step - polymerase extends from primer
repeat 1 -3

Question 4

methods to discriminate between transcriptional and pos transcriptional gene expression >

Answer 4

inhibition of rna pol 2 - DRB
run-on transcriptional analysis - ISolate nuclei and incubate them with radioactively labelled nucleotides
b- isolate rna and use it to probe a membrane containing gene of interest

Question 5

how are transcriptional control elements identified

Answer 5

test the specific effects of dna sequences of transcritpion
reporter genes - eassily assayed gene products
x-gal - blue colour
luciferin - light emission

Question 6

deletion analysis of control elements

Answer 6

pcr fragments of different lenths from promoter
insert it into plasmid vector with a reporter gene
ligate vector to carry reporter
transform e.coli and isolate plasmid DNAs

transfect each plasmid into cell

prep cell extract and assay activity of reporter enzyme

Question 7

in vivo - does TF x affect expression of reporter gene in cell

Answer 7

insert two plasmids into nucleus one with a reporter gene and x binding site and another with just gene for TF x

if x does affect expression we will see reporter-gene transcripts

Question 8

application of reporter assays

Answer 8

define promoters
compare promoter activity in different cells
define functional parts of promoters
identify regulatory elements
study effect of other expressed proteins
measure TF activity

Question 9

how are TF identified

Answer 9

Databases for known dna binding sites
DNA binding assays
-DNase 1 footprinting
gel mobility shift
chromatin immunoprecipitation

purification - dna affinity columns

Question 10

dnase 1 footprinting

Answer 10

dna labeled on one strand with protein of intrest
complex trreated with DNase 1 which cleaves dna not masked by TF
control DNA sample is treated und3er same conditions without TF

finally banding patters of two are compared to locate footprint region where TF has shielded DNA

Question 11

EMSA

Answer 11

incubate radioactive dna probe with protein and electropherese under non denaturing conditions

Question 12

CHIP

Answer 12

Purify DNA-bound complexes - after cross linking and shearing
PCR with gene specific primers