BM3502 Neuroscience
& Neuropharmacology
Data Handling
Practical
Student Answer
Booklet
Student Name:…………………………………………………………………………..
Please do not lose this booklet or forget to bring it with you to your
practical sessions. The practical takes place in two phases spread
over two weeks. The first week will involve 3 data handling
exercises introducing you to some commonly-used statistical tests,
and how you might use them to analyse specific types of data. In
the second week, you will be given further tuition in data handling,
but this will be followed by an exercise that you will work through
using the skills you have learned over the past 2 weeks. You will
write these answers into your sheet and submit them for marking.
This exercise forms part of your continuous assessment for this
course.
If you do not understand what you are meant to do, or if you have
any problems at all, please ask a demonstrator or the course
organiser, Dr Derek Scott, who will be present at all of the sessions.
1
Introduction
Log on to a PC. You may work in pairs for this exercise if there are
not enough PC’s. Go to the Desktop folder for “Life Sciences &
Medicine”, then open “Medical Sciences”, then “Biomedical
Sciences”. Open “Prism 5”. DO NOT USE Prism 3 or 4 – you will not
all be able to access the program, and the layout is slightly different
to the instructions given in this handout.
Open the “BM3502” folder within the “Biomedical Sciences” folder,
and open the “Stats1.xls” file. This contains the experimental data
that you will analyse with Prism.
Read through each exercise and check if hints are given in each
section that tell you how to use the software. If in doubt, ask for
help!
Prism 5 is a handy program that not only analyses data, but also
plots graphs. You can use Prism 5 to write your lab reports or
Honours thesis if you want to get more professional results than
those produced by Microsoft Excel.
Statistics is not always everyone’s favourite topic in science, but it
is essential that you understand some of the basics so you can
judge whether data is valuable or not.
2
Week 1 - Data Handling Exercise 1: Student’s t-test.
How do I set up my Prism worksheet?
NB refer to Prism “Help, Help me to decide”, “INTERPRETATION” or
“Find” for answers to some questions).
Open Prism 5
Select the tab labelled “Column”
Make sure the screen looks like this:
Now click on the “Create” button.
What to do with the data:
Paste the data you want to analyse from Excel into first two Y
columns (e.g. Time zero and +100 min columns for Drug A data)
label columns (‘Drug A Time zero’ and ‘Drug A +100 min’)
To avoid confusion make sure you label each data column.
3
To perform the data analysis: click on:
(1) Analyse, then OK then
(2) Column Analyses, then
(3) t test (you need to decide which kind of t test to use and to pick
appropriate P value options), then OK
Record Results and look at data on Graph
Repeat above procedure for comparing other columns of data
What do I analyse?
1. Groups of mice were injected with a hypothermic agent (Drug A)
or its vehicle (Saline) at time zero. The rectal temperature of each
animal was measured just before the injections were made (time
zero) and at +100 min (the time of maximum hypothermia). Make
use of Prism to determine whether:
(a) Drug A produced a statistically significant decrease in rectal
temperature (Student’s t test; P < 0.05):
answer: yes no (P = __________ )
(b) The mean rectal temperatures of the two groups at time zero
are significantly different (Student’s t test;
P < 0.05): answer: yes no (P = __________ )
(c) The mean rectal temperatures of the two groups at +100 min
are significantly different (Student’s t test;
4
P < 0.05): answer: yes
no
SALINE
Mouse
(P = __________ )
DRUG A (1 mg/kg i.p.)
Rectal temperature
(oC)
At time
At time
+100
zero
min
Mouse
Rectal temperature
(oC)
At time
At time
+100
zero
min
1
38.0
37.9
7
38.0
28.4
2
37.9
36.7
8
38.0
28.6
3
38.7
37.7
9
38.1
27.9
4
37.6
37.7
10
38.0
27.7
5
38.2
38.0
11
37.4
27.0
6
37.2
36.9
12
37.6
27.4
2.
What types of t test did you use for each calculation and why?
(circle correct answers)
(a) (i) paired or unpaired? (ii) 1-tailed or 2-tailed?
(b) (i) paired or unpaired? (ii) 1-tailed or 2-tailed?
(c) (i) paired or unpaired? (ii) 1-tailed or 2-tailed?
3.
What does the P value tell you? (search for “P value” in Prism
Help)
4.
What is meant by the term “Null hypothesis”?
5.
What assumptions have you made about the data when
applying the t test(s) you used?
(search for “t test or non-parametric test” in Prism Help)
5
6.
What is the difference between standard deviation and
standard error of the mean?
(search for “SD” in Prism Help).
7.
What do you understand by the term “confidence interval”?
(search for “Confidence” in Prism Help).
8.
Are any statistically significant differences that you detected
“scientifically trivial”? (This means are there values so close to
0.05 that you are not sure there really is a difference or not?)
6
Week 1 - Data Handling Exercise 2: Non-parametric tests.
Note: you will need to work out from Prism what you use
Mann-Whitney U-tests and Wilcoxon’s tests for i.e. paired or
unpaired data.
1. Groups of mice were injected with a hypothermic agent (Drug B)
or its vehicle (Saline) at time zero. The rectal temperature of each
animal was measured just before the injections were made (time
zero) and at +100 min (the time of maximum hypothermia). Make
use of Prism to determine whether:
(a) Drug B produced a statistically significant decrease in rectal
temperature (Mann-Whitney U-test or Wilcoxon’s test; P < 0.05):
answer: yes no (P = __________ )
(circle yes or no AND write the P value on the line)
(b) The mean rectal temperatures of the two groups at time zero
are significantly different (Mann-Whitney U-test or Wilcoxon’s test;
P < 0.05): answer: yes no (P = __________ )
(c) The mean rectal temperatures of the two groups at +100 min
are significantly different (Mann-Whitney U-test or Wilcoxon’s test;
P < 0.05): answer: yes no (P = __________ )
SALINE
Mouse
DRUG B (10 mg/kg i.p.)
Rectal temperature
(oC)
At time
At time
+100
zero
min
Mouse
Rectal temperature
(oC)
At time
At time
+100
zero
min
1
38.1
38.0
7
38.1
30.9
2
37.7
36.7
8
38.2
31.0
3
38.6
37.6
9
37.5
29.5
4
37.5
37.8
10
37.4
29.2
5
38.1
38.2
11
38.1
30.2
6
37.5
37.3
12
37.9
29.9
7
2.
What types of test did you use for each calculation and why?
(circle test used)
(a) Mann-Whitney U-test or Wilcoxon’s test?
(b) Mann-Whitney U-test or Wilcoxon’s test?
(c) Mann-Whitney U-test or Wilcoxon’s test?
3.
What assumptions have you made about the data when
applying the test(s) you used?
(search for “Mann” and “Wilcoxon” in Prism Help)
4.
What are the advantages of these tests over Student’s t test?
Hint: make use of Prism Help.
8
Week 1 - Data Handling exercise 3: ANOVA plus post-hoc
tests for multiple comparisons.
1. Groups of mice were injected with two hypothermic agents
(Drugs A and B) or its vehicle (Saline) at time zero. The rectal
temperature of each animal was measured just before the injections
were made (time zero) and at +100 min (the time of maximum
hypothermia). Make use of Prism to determine whether:
(a) Drugs A and B produced decreases in rectal temperature that
were significantly greater than the decrease that followed the
injection of saline (ANOVA + Dunnett’s test; P < 0.05): answers:
Saline vs. Drug A:
Saline vs Drug B:
yes
yes
no
no
(P = __________ )
(P = __________ )
(b) Drugs A and B produced statistically significant decreases in
rectal temperature that were significantly different from each other
and/or from the decrease that followed the injection of saline
(ANOVA + Bonferroni’s test; P < 0.05): answers:
Saline vs. Drug A:
Saline vs Drug B:
Drug A vs Drug B:
SALINE
2.
Mouse
Rectal
temperature
(oC) at time
+100 min
1
yes
yes
yes
no
no
no
(P = __________ )
(P = __________ )
(P = __________ )
DRUG A (1 mg/kg
i.p.)
DRUG B (10
mg/kg i.p.)
Mouse
Rectal
temperature
(oC) at time
+100 min
Mouse
Rectal
temperature
(oC) at time
+100 min
38.0
7
28.3
13
30.8
2
36.9
8
28.5
14
31.1
3
37.5
9
27.8
15
29.6
4
37.9
10
27.6
16
29.1
5
38.3
11
27.1
17
30.1
6
37.4
12
27.5
18
30.0
What assumptions have you made about the data when
applying the tests you used?
9
(search for “ANOVA” in Prism Help)
3.
Could you have used a non-parametric test instead (e.g. the
Kruskal-Wallace test)?
Why might you have decided to do this?
(search for “Kruskal” in Prism Help)
Hint: make use of Prism Help.
See “Which post test” in Prism for information about Dunnett and
Bonferroni tests.
10
Week 2 – Data Handling Exercise 1: Problem Solving – DoseResponse Curves.
In experiments with the guinea-pig isolated small intestine it was
found that Drug X can inhibit electrically-evoked contractions of this
tissue at concentrations that do not affect its responsiveness to
acetylcholine (ACh). The inhibitory effect of drug X on the amplitude
of the electrically-evoked contractions was concentrationdependent. Further experiments showed drug X to be susceptible to
antagonism by 10 nM SR141716A, a selective antagonist of
cannabinoid CB1 receptors (see below). The KB value of SR141716A
against the CB1 cannabinoid receptor agonist, CP55940, in this
tissue was found to be 0.99 ± 0.06 nM (mean ± s.e.; n=6) when
the measured response was CP55940-induced inhibition of
electrically-evoked contractions (KB = dissociation equilibrium
contant). The degree of inhibition of evoked contractions induced by
Drug X was calculated in percentage terms (%inhibition) by
comparing the amplitude of the twitch response after each addition
of Drug X with its amplitude immediately before the first addition of
this agonist. Each log concentration-response curve of X was
constructed using a different strip of tissue. CB1 receptor agonists
are known to act though presynaptic CB1 receptors in guinea-pig
small intestine to inhibit electrically-evoked release of the
contractile transmitter, acetylcholine, and so decrease the
amplitude of evoked contractions.
What to do:
Open Prism and the Stats2.xls file from the BM3502 folder.
Select the XY tab:
X column: you don’t have to do anything.
Y column: 6 replicates for Exercise 1 (Table 1 of Excel Sheet 1) and
“plot a single Y value for each point” for Exercise 2 (Table 2 of Excel
Sheet 1).
Data:
paste concentration data into X (concentrations) and first twelve Y
columns (two sets of % inhibition data)
To avoid confusion make sure you label each data column.
Data analysis: click on:
(1) Analyse, then
(2) look for “XY analyses” then
(3) Nonlinear regression (curve fit) then OK then
(4) Classic equations from prior versions of Prism, then
(5) Sigmoidal dose-response (variable slope) then OK
11
Record Results and also look at data on Graph
Hint: make use of PRISM Help
(1) Use Prism and the data in Excel Sheet 1 to answer the following
questions: show units for all your data.
Use nanomolar (nM) as your concentration units (1 nM = 10-9 M).
(a) what is the EC50 of Drug X
(i) in the absence of SR141716A?
(ii) in the presence of 10 nM SR141716A?
__________
__________
(b) what is the 95% confidence interval for the mean EC50 of X
(i) in the absence of SR141716A?
__________ to _____________
(ii) in the presence of 10 nM SR141716A?
__________ to _____________
(c) does SR141716A produce a statistically significant dextral shift
in the log concentration-response curve of X? yes or no
(d) what is the 95% confidence interval for the slope of the log
concentration-response curve of X
(i) in the absence of SR141716A?
_____________ to _____________
(ii) in the presence of 10 nM SR141716A?
_____________ to _____________
(e) does the dextral shift deviate significantly from parallelism?
yes or no
______________________________________________________
(d) what is the 95% confidence interval for the Emax of X
(i) in the absence of SR141716A?
_____________ to _____________
(ii) in the presence of 10 nM SR141716A?
_____________ to _____________
(e) does SR141716A produce a statistically significant change in the
Emax of X? (Emax is also known as TOP on Prism i.e. the maximum
response)
yes or no
P value = _____________
12
(f) what is the mean concentration ratio (see below) in this set of
experiments?
concentration ratio = _____________
Note: The concentration ratio is the EC50 value for the Drug
X in the presence of the antagonist divided by the EC50 of
Drug X alone.
(g) use the Schild equation (see below) to calculate the mean KB
value of SR141716A against Drug X.
KB = ________________
*Schild equation:
KB = (SR141716A concentration)/(concentration ratio-1)
13
(2) Select “New Table” in Prism and change to “Enter and plot a
single Y value for each point” for Y column on the startup menu.
Then, using the data in Table 2 of Excel Sheet 1 (same data as in
Table 1 but arranged differently) and Prism calculate individual
EC50 values for each experiment (experiments 1 to 6). Paste these
EC50 values into Table 3 of Excel Sheet 1. These should reveal
individual KB values for SR141716A against X in Table 4 of Excel
Sheet 1 (calculated using the Schild equation)*.
Is the mean KB value of SR141716A against Drug X significantly
different from that of SR141716A against CP55940? (use the two
sets of values in Excel Table 4).
(i) yes or no
(ii) P value = _________
(iii) †statistical test used =
(iv) What is the main assumption made by the Schild equation
(about SR141716A)?
*Schild equation:
KB = (SR141716A concentration)/(concentration ratio-1)
†Each log concentration-response curve of X was
constructed using a different strip of tissue.
NB Means can be calculated from columns of data using
“Column Statistics” in “Statistical Analyses” of Prism (use Y
columns for your data having changed “Column Format” of X
column to “None”).
14
Week 2: Problem Solving Exercise 2 – ASSESSED EXERCISE
You will not receive tuition during this exercise, but we will provide
assistance with basic function of GraphPad Prism 5. Use the skills
and notes you have made over the past two weeks to help you
solve the problems. You must submit your answers either before
you leave the practical session or soon after by the deadline stated
in the course manual, and these will be marked. This will form part
of your continuous assessment for this course.
Problem 2
In experiments with the guinea-pig isolated small intestine it was
found that Drugs X and Y can inhibit electrically-evoked contractions
of this tissue at concentrations that do not affect its responsiveness
to acetylcholine (ACh). The inhibitory effects of both drugs on the
amplitude
of
the
electrically-evoked
contractions
were
concentration-dependent. SR141716A (10 nM) produced parallel
dextral shifts in the log concentration response curves of Drugs X
and Y in this tissue. SR141716A (10 nM) also produced a parallel
dextral shift in the log concentration response curve of CP55940
when the measured response was CP55940-induced inhibition of
electrically-evoked contractions. You may assume that Drug X is
the drug that featured in Problem I. The degree of inhibition of
evoked contractions induced by Drug X or Y was calculated in
percentage terms (%inhibition) by comparing the amplitude of the
twitch response after each addition of X or Y with its amplitude
immediately before the first addition of X or Y. For each drug, each
log concentration-response curve was constructed using a different
strip of tissue.
Use Prism (Results & Graph) & the data in Excel Sheet 2 Tables 1*
& 2** to answer the following questions. Show units for all your
data. Use nanomolar (nM) as your concentration units (1 nM = 10-9
M).
______________________________________________________
(1a) what are the EC50 values of X and Y and their 95% confidence
intervals (in brackets)*?
*EC50 of X = _____________ ( ___________and ___________ )
*EC50 of Y = _____________ ( ___________and ___________ )
______________________________________________________
(1b) are the EC50 values of X and Y significantly different?
yes or no
______________________________________________________
(2a) what are the Emax values of X and Y and their 95% confidence
intervals (in brackets)?
*Emax of X = _____________ ( ___________and ___________ )
*Emax of Y = _____________ ( ___________and ___________ )
15
______________________________________________________
(2b) are the Emax values of X and Y significantly different?
yes or no
______________________________________________________
(3a) assuming the Schild equation is valid for the data, what are the
KB values of SR141716A against X, Y & CP55940?
**mean KB (10 nM SR141716A against Drug X) =
_______________
**mean KB (10 nM SR141716A against Drug Y) =
_______________
**mean KB (10 nM SR141716A against CP55940) =
_______________
Schild equation: KB = (SR141716A concentration)/(concentration
ratio-1)
______________________________________________________
(3b) are the KB values of SR141716A against Drug X, Drug Y and
CP55940 significantly different?
(i) yes or no (Drug X vs. Drug Y): P value = _______________
(ii) yes or no (Drug X vs. CP55940): P value = _______________
(iii) yes or no (Drug Y vs. CP55940): P value = _______________
(iv) statistical test(s) used =
______________________________________________________
(v) “post-test” (if any) =
______________________________________________________
4. How should Drugs X and Y be classified? Give reasons for your
answer (maximum of 100 words). (Hint – look at the curves on the
graphs!)
16
5. Does the ratio of the EC50 values of Drugs X and Y provide a
useful measure of their relative potency?
Give reasons for your answer (maximum of 100 words).
Hint: Prism defines an EC50 value as the concentration of a drug
that produces an effect that is half the size of the effect produced
by a maximal concentration of the same drug.
17