Muscle analysis

Question 1

How can muscle analyses be carried out?

Answer 1

Blood samples; muscle biopsies; magnetic resonance imagery; near-infrared spectroscopy; nerve and muscle stimulation coupled with EMG; dual-energy X-ray absorptiometry; cadaver dissection

Question 2

How can blood samples be used to analyse muscles?

Answer 2

Cells contain genetic info; plasma contains metabolites and hormones etc; can use single blood samples or repeated via a cannula

Question 3

What are the four stages required in a muscle biopsy?

Answer 3

Incision –> needle –> extract –> analyse

Question 4

What three molecule types need to be considered when analysing muscles?

Answer 4

DNA, mRNA, and proteins

Question 5

How can DNA be analysed?

Answer 5

PCR, then gel electrophoresis

Question 6

What is the process of PCR?

Answer 6

Centrifuge; target specific gene; denature at 95 Celsius; ssDNA result; anneal primers onto ssDNA at 40-60 Celsius; primers from complementary base pairs with ends of ssDNA; Taq DNA polymerase extend chain with free nucleotides

Question 7

How many times is PCR repeated, and how many DNA replications can result?

Answer 7

4 - 30 times; forms between 2^4 - 2^30 replications

Question 8

What are the benefits of PCR?

Answer 8

Allows analysis of genes present in genome; is sensitive; amplifies; can source from blood or tissue

Question 9

How can mRNA be analysed?

Answer 9

RT-PCR, then gel electrophoresis

Question 10

What is the process of RT-PCR?

Answer 10

Isolate mRNA from tissue or cell; add primer to region of interest; reverse transcriptase starts at poly-A end and forms DNA from mRNA; PCR

Question 11

What are the benefits of RT-PCR?

Answer 11

Allows analysis of expressed genes; mRNA changes whilst DNA doesn’t (mRNA will not contain introns/ other info not needed as proteins are not needed)

Question 12

How can proteins be analysed?

Answer 12

Proteomics, blotting, mass spec, and histology

Question 13

What is the process of proteomics?

Answer 13

1-D electrophoresis, proteins separated on an acrylamide gel: gel is injected by polyampholytes; pH gradient is established; voltage is applied; proteins will stop in a position where the pH is equal to the isoelectric point of the protein, as valence will be equal to 0. 2-D electrophoresis for more complex proteins such as myosin and actin: proteins in IPG (immobilised pH gradient) strip migrate into polyacrylamide gel during SDS-PAGE; separates based on size; each protein species appears as a spot on the gel when detected

Question 14

How are the proteins with contractile proteins separated?

Answer 14

Heavy (200kDa) and light (16-27kDa) chains of myosin separated; actin (43kDa) causes smear on gels due to high protein content, so need to undergo further sample preparation to separate isoforms

Question 15

What are the benefits of proteomics?

Answer 15

Allows analysis of proteins expressed by a genome or altered by post-translation modifications; shows if protein synthesis is up- or down-regulated as a whole; proteome changes, whilst DNA doesn’t; allows analysis of genetic and environmental factors in health and disease

Question 16

What is the qualitative measure given by proteomics?

Answer 16

Upregulation of protein expression

Question 17

What is the quantitative measure given by proteomics?

Answer 17

Measure of specific protein change

Question 18

What is the process of blotting?

Answer 18

After electrophoresis, proteins blotted onto a membrane; specific primary antibody binds to protein; secondary antibody binds to primary antibody; substrate binds to secondary antibody; substrate/enzyme is activated and a colour reaction occurs’ colour reaction is proportional to amount of protein, given a quantitative measure of level of expression; detection of secondary antibody gives info about mass of protein (quantitative) and post-translational changes (qualitative)

Question 19

What are the benefits of blotting?

Answer 19

Identifies specific protein

Question 20

What is the process of mass spec?

Answer 20

Electrons fired; breaks into ions; ion fragments have different masses and charges; detected in different locations; protein identified by m/z ratios of fragments

Question 21

What is the benefit of MS?

Answer 21

Quantifies how much a specific protein has been expressed within a cell

Question 22

What is the process of immunohistology?

Answer 22

Primary antibody binds to protein; secondary antibody binds to primary antibody; secondary protein is attached to a fluorescent tag; tag detected with fluorescence microscope; specific proteins are assessed

Question 23

What are the benefits of histology?

Answer 23

Allow the study of protein location; allows muscle fibre architecture to observed by cutting sections of muscle

Question 24

What is muscle plasticity?

Answer 24

Changes in gene expression and protein synthesis are related to contractile demand

Question 25

What are two examples of muscle plasticity?

Answer 25

Fibre type switch; hypertrophy

Question 26

How can muscle fibre types be detected?

Answer 26

ATPase staining; myosin staining; glycogen staining; IMTG staining; electron microscopy

Question 27

What fibres would have highest glycogen content?

Answer 27

Type IIx

Question 28

What fibres would have highest IMTG?

Answer 28

Type I

Question 29

How is IMTG stained?

Answer 29

With a dve that attaches to lipids