RNA Methods

Question 1

What is Western Blotting Gel Made Of

Answer 1

Polyacrylamide. (Polyacrylamide Gel Electrophoresis)

Question 2

Purpose of Sodium Dodecyl Sulfate in Gel Electrophoresis

Answer 2

Denatures Protein

Question 3

Purpose of Eastern Blotting

Answer 3

Epigenetic Marker separation

Question 4

Purpose of Northern Blocking

Answer 4

RNA separation

Question 5

Purpose of southern Blocking

Answer 5

DNA separation (nucleic acid probe)

Question 6

Purpose of Western Blocking

Answer 6

Protein separation (antibody probes)

Question 7

Western Blotting Steps

Answer 7

Protein Electrophoresis (separation of protein on polyacrymalide gel based on size + charge), trsnfer separated proteins onto membrane, run Western Blotting

Question 8

Separated protein on membrane Layout (bottom to Top)

Answer 8

Anode, 2 filter paper, membrane, polyacrymalide gel, 2 filter paper and cathode

Question 9

Western blotting Stage Steps

Answer 9

Viris isolation + lysis, protein suspension, SDS-page, electrotransfer (PVDF membrane), antibody probing and chemi-imaging

Question 10

The importance of having a selective antibody in Western Blotting

Answer 10

Non-selective antibodies bind to proteins in sample other then target. Creates background noise reducing accuracy of readings

Question 11

Beta Actin Outline

Answer 11

Western Blotting control (reference). Present in almost all eukaryotic cells to the same level, stable across all conditions. Protein of interest concs are inferred by comparison

Question 12

ELISA Vs Western Blotting

Answer 12

ELISA; rapid (no separation), better quantification. Western; protein ID

Question 13

Enzyme Linked Immunosorbent Assay Methods

Answer 13

Antigen is immobilised on a solid surface. Complex formed with an antibody linked to a reporter enzyme. 2 types; direct (1 antibody) and indirect (2 antibodies)

Question 14

Transcriptome Outline

Answer 14

Full range of mRNA expressed in an organism’s system or specific cell types. Transcriptomes can be compared to identify gene expression

Question 15

Why is mRNA often converted to cDNA

Answer 15

DNA is less reactive (susceptible to degradation) then RNA. DNA doesn’t have exposed nitrogen bases and ribose sugar is more reactive then deoxy (as ribose has OH)

Question 16

Purpose of studying RNA

Answer 16

Gene expression analysis. Help cancer diagnosis; tumour type, metastasis risk and effective treatment strategy

Question 17

How is mRNA converted to cDNA

Answer 17

reverse transcriptase acts on oligonucleotide primer to form a complementary second strand

Question 18

Absolute Quantification Using qPCR

Answer 18

Create a standard curve (+ equation) with a samples of known DNA concentrations. DNA concentration of unknown sample is evaluated from equation

Question 19

Transcriptome Analysis Outline

Answer 19

Insights into patterns of gene expression; understand cell/tissue dev (amy change from baseline due to disease/aging). As gene concentrations don’t change independently, if 1 gene is overexpressed another may be under expressed. HAve to use cells from tissue of interest as gene expression is different in cells across differnt parts of body

Question 20

RNA Sequencing Application Outline

Answer 20

Gene expression analysis, alternative splicing analysis and non-coding RNA profiling

Question 21

DNA Sequencing Outline

Answer 21

Used for genome sequencing, genetic mutation ID, genetic diversity and forensic analysis. DNA sequencing of a genome doesn’t differ between cell types

Question 22

RNA Sequencing Outline

Answer 22

Provides insight into gene expression and regulation. Measures gene expression levels, ID different expressed genes. RNA sequences vary from person to person and cell type to cell type within the same person