Research Techniques

Question 1

What is a northern blot used to identify?

Answer 1

Used to detect specific RNA from a mixture of RNA

Question 2

Technique of northern blot

Answer 2

1. Denature the RNA within the sample into single stranded (no bonding present)
2. Separation according to size using gel electrophoresis
3. Transfer onto blotting membrane
4. Add probe complementary to RNA sequence wanting to be identified (incl. radioactive/fluorescent label)
5. Identifies RNA of interest

Question 3

What denaturing agent is used in northern blotting?

Answer 3

formaldehyde or DMSO

Question 4

How does a gene get to a protein?

Answer 4

gene (DNA) is transcribed into RNA. RNA is translated into protein

Question 5

Are RNA molecules positively to negative changed?

Answer 5

negatively charged

so move from negative to positive electrode during gel electrophoresis

Question 6

What is PCR used for?

Answer 6

make copies a specific DNA region in vitro

Question 7

What are the three main stages of PCR

Answer 7

1. Denaturing - double stranded DNA is heated and separated into two single strands (96 degrees)
2. Annealing - temperature is lowers to enable DNA primers to attach to template DNA (55 degrees)
3. Temperature is raised and DNA polymerase added to make new DNA piece

Question 8

What is RT-PCR used for?

Answer 8

Reverse-transcriptase PCR. use reverse transcriptase to convert RNA to cDNA then amplify these specific DNA targets using PCR

Question 9

What is reverse transcriptase?

Answer 9

Enzyme used to generate complementary DNA from RNA template

Question 10

What is immunohistochemistry

Answer 10

Technique that used antibodies conjugated to enzymes/fluorescent dyes that catalyse reaction to form detectable compounds to visualise and identify specific antigens in a tissue sample

Question 11

What biological concept does IHC capitalise on?

Answer 11

Antigen-antibody specificity

Question 12

Overview of IHC technique using HRP

Answer 12

1. Tissue section is fixed and stained using a primary antibody which is specific to the target protein, after sufficient time allowed for the antibody to bind, excess primary antibody is washed away
2. Secondary antibody is added, which carries linker molecule with horseradish peroxide (HRP) enzymes, which bind to the primary antibody after sufficient time and then excess is washed away
3. DAB is added which HRP transforms into a brown precipitate which allows easier visualisation of the protein of interest

Question 13

What is siRNA interference screening used for?

Answer 13

Interfere with the translation of proteins by binding to and promoting the degradation of mRNA at specific sequences

Question 14

Structure of siRNAs

Answer 14

21 nucleotides long with 3’ overhangs at each end

Have specificity to match mRNA of what’s being investigated

Question 15

Controls for using siRNAs

Answer 15

not 100% efficient, not the same of KO

need to check changes in protein/mRNA levels after - through RTPCR or antibody experiment

Question 16

What does patch clamp electrophysiology measure?

Answer 16

Records ionic currents that flow across a cell surface. Usually transmembrane voltage is control to just measure current

Question 17

What is immunoprecipitation?

Answer 17

using an antibody to purify a specific target (protein)

Question 18

What is coimmunoprecipitation?

Answer 18

used to identify protein-protein interactions by precipitating out complexes made up of different proteins

Question 19

What is Southern blotting used to separate?

Answer 19

Used in the identification of a particular size of DNA in a mixture of other similar molecules

Question 20

What is northern blotting used for?

Answer 20

detection and analysis of proteins

Question 21

Technique of western blotting

Answer 21

1. protein separated by gel electrophoresis
2. gel transferred onto membrane (e.g nitroceullose)
3. add antibodies for the proteins you want to detect (can use primary and secondary)

Question 22

Why would epitope tagging be used?

Answer 22

No antibodies available for proteins that wants to be detected

Question 23

How does epitope tagging work?

Answer 23

Epitope tags are small peptide sequences for which cheap, high affinity primary antibodies are available for. These tags can be engineered at any site in recombinant cDNA to introduce an antibody binding epitope
Then go on to use western blot, immunohistochemistry and immunoprecipitation