Stats workshop.

You're going to calculate an ANOVA and

demonstrator FINISH THIS SHEET FOR
These numbers show the maximum drop (ie. minimum value) in intracellular
extracellular calcium (20mM) over the 30s post ca
Group 1 Group 2
AS syndrome 20mM calcium NHMC 20mM calcium
1.119186 0.978445 0.867681
1.016641 0.929748 0.394444
1.308806 1.035457 0.758896
1.153704 1.014493 1.036364
1.107556 1.013928 1.251154
1.18125 1.003036 0.832194
0.830335 0.982739 0.590909
0.972752 1.008749 0.444892
1.009654 0.969035 0.982703
0.968504 0.988436 1.01615
0.993214 0.949153 1.037234
1.011789 0.958474 0.936599
0.983837 1.011352 0.958231
0.993135 1.169666 0.950593
1.220221 1.001806
This graph shows NHMC data over time (as measured in the 1.415456 0.900916
red circle within the green cell) . Note the dip (the MINIMUM
response) before the increase in intracellular calcium. We're 1.557653 1.049938
looking to see if all THREE cell lines show tha similar dip in 1.011927
intracellular calcium, just before the elevation.
1.15895
1.139368

TO GET THE TOTAL SSQ

0.031786 0.00141 0.005361
ANOVA CALCULATIONS show where in a population the 0.005737 0.000124 0.298614
variation lies: is it BETWEEN GROUPS (=treatment) OR is 0.135354 0.008941 0.033126
it WITHIN THE GROUPS (RESIDUAL). TO FIGURE THIS 0.045285 0.005416 0.009113
OUT WE FIRST MEASURE THE TOTAL VARIATION 0.027774 0.005333 0.096257
(starting with the SSQ). We then subtract the WITHIN 0.057768 0.003861 0.011817
GROUP (RESIDUAL) VARIATION (SSQ) to generate a 0.012225 0.00175 0.122494
between group (treatment) variation. Unlike the t-test 0.001015 0.004603 0.246025
which effectively measures difference between means 0.004727 0.000792 0.001747
over variance of the samples, the ANOVA calculates the
0.000762 0.00226 0.005662
(F) ratio of between groups/within group variations
0.002737 6.81E-05 0.00928
(SSQs). We then compare that ratio value to a
0.005025 0.000309 1.85E-05
hypothetical F value (from an F probability distribution
0.001844 0.004963 0.0003
calculated for almost every number of degrees of
0.002728 0.052334 9.39E-05
freedom). If your F value is large it means that the
major variability arises between groups, rather than 0.07802 0.003709
within them. Thus, a F value larger than the 0.225203 0.001599
"hypothetical " F distribution value (at a given number
of degrees of freedom and a particular level of
significance (say 0.05)) means that the source of
 calculated for almost every number of degrees of
freedom). If your F value is large it means that the
major variability arises between groups, rather than
within them. Thus, a F value larger than the
"hypothetical " F distribution value (at a given number 0.380384 0.011889
of degrees of freedom and a particular level of 0.005045
significance (say 0.05)) means that the source of 0.047545
variability bewteeen groups is unlikely to appear by 0.039389
chance!!!! You can now REJECT the null hypothesis!!
What was the null hypothesis, again??

0.82649 0.882643 0.875234

TOTAL SSQ 2.944437 2.944437

DF 135 135 Why "-1"?

the value below =AVERAGE(C6:D27)

within Group averages 1.059884 0.913069
within GROUP SSQ 0.003517 0.006632 0.00206
0.00187 0.016935 0.268971
0.061962 0.000597 0.023769
0.008802 0.00206 0.015202
0.002273 0.002112 0.114301
0.01473 0.003232 0.006541
0.052693 0.005951 0.103787
0.007592 0.002615 0.21919
0.002523 0.008254 0.004849
0.00835 0.005105 0.010626
0.004445 0.012261 0.015417
0.002313 0.010284 0.000554
0.005783 0.002355 0.00204
0.004455 0.012052 0.001408
0.025708 0.007874
0.126432 0.000148
0.247774 0.018733
0.009773
0.060457
0.051211

Residual (within group) SSQ 2.368249 2.368249

Residual (within group) df 133 133

SSQ df MS
total 2.944437 135 0.021811
residual within group 2.368249 133 0.017806

Treatment (between group) 0.576188 2 0.288094 16.17926

 if F =

This table is a little backwards, we're looking for 0.95 (p = 0.05), but is our
e an ANOVA and maybe a t-test by hand! At the end of the session s
THIS SHEET FOR 66%. FINISH SHEET TWO and you can have the mis
rop (ie. minimum value) in intracellular calcium in response to the application of
ar calcium (20mM) over the 30s post calcium addition
Group 3
NHMC 20mM calcium HIP 20mM calcium
START HERE! A podocyte experiment. I'm measuring calc
1.050713 0.956925 0.820309 0.893642
sets of podocytes from:
1.009025 0.953333 0.908686 1.273095 (1) A person with Alport syndrome (AS; they cannot make
0.98279 0.952381 0.813294 0.776322 (2) A person with healthy kidneys (NHMC)
0.970149 0.967908 0.772358 0.874025 (3) A "podocyte" cell line that has been immortalized with a
1.055195 0.959524 0.780976 0.917197
1.030877 0.975201 0.742268 0.830821
0.954302 0.949675 0.997188 0.856549 The podocytes have been loaded with a dye (FURA2
0.957895 0.963512 0.877963 0.930351 measurements (left, with the blue background) show intrac
0.987246 0.944614 0.8654 0.850498 simplify the experiment I've divided each value by the resp
0.998172 0.96688 0.868651 0.811725 responses in the 30 seconds AFTER calcium addition!
0.973502 0.965347 0.894323 0.776618
0.928571 0.98791 0.778116 0.787746 Low fluorescence numbers means low intracellular
experiment is simple, I've added extracellular calcium and
0.792572 0.892229 0.961798 1.090675
receptor triggers an elevation of intracellular calcium in res
0.779307 0.931129 0.903226 0.811174
intracellular calcium goes up in NHMC, it drops!!
0.841949 0.953333 0.970549 1.049847
0.727717 0.852733 0.994819 0.88453 My question is: Do all three kinds of podocytes (AS, NHMC,
0.827199 0.765616 1.053319 0.955474
0.813913 0.742313 0.8724 0.974576 (1) How should we analyse it? Here are your choices; t
0.734062 0.819484 0.886022 0.904348 (2) Analyse it, showing your working.
0.775754 0.869101 0.830754 0.921747
0.816176 0.942253 0.894903 THREE GROUPS OF NUMBERS
CONTINUAL DISTRIBUTION OF NUMBERS
0.893642 0.914714
REALLY ONLY ONE LOGICAL CHOICE OF TEST.............

HE TOTAL SSQ
0.012059 0.000257 0.014542 0.002233
0.004641 0.000155 0.001038 0.110353
0.001755 0.000132 0.016283 0.027086
0.000855 0.000729 0.028407 0.004472
0.013063 0.000347 0.025576 0.000562
0.008096 0.001177 0.039455 0.012118
0.00018 7.7E-05 0.003168 0.007115
0.000289 0.000511 0.003961 0.000111
0.002148 1.38E-05 0.0057 0.008173
0.00328 0.000675 0.00522 0.016686
0.001063 0.000598 0.002169 0.026989
0.000152 0.00221 0.026499 0.023456
0.022001 0.002369 0.000437 0.022432
0.026112 9.55E-05 0.001419 0.016829
0.009791 0.000155 0.000879 0.011869
0.045447 0.007774 0.002907 0.003178
0.012928 0.030725 0.012638 0.000212
0.016126 0.039437 0.004692 0.001134
0.042782 0.014742 0.003012 0.001336
0.027273 0.005155 0.012132 0.000367
0.015556 1.83E-06 0.002116
0.002233 0.000686

0.862747 0.884811 0.858139 0.881096

make sure that these averages belong to the correct cells!

0.895657 Basically we're taking the SSQ/d
0.018946 0.001923 0.005677 4.06E-06 mean square, MSQ)) and subtra
0.009208 0.001621 0.00017 0.14246 each gro
0.004861 0.001545 0.006784 0.014241
0.003258 0.003007 0.015203 0.000468
0.0202 0.002158 0.013152 0.000464
0.013879 0.00386 0.023528 0.004204
0.0017 0.00134 0.010309 0.001529 What we're left with is the variab
0.002009 0.002545 0.000313 0.001204 then compare our variability with
a bit like a Gaussian, but is skew
0.005502 0.000995 0.000915 0.002039
greater than the "predicted" dis
0.007243 0.002896 0.000729 0.007045
then we conclude that such a big
0.003652 0.002733 1.78E-06 0.01417
rejec
0.00024 0.005601 0.013816 0.011645
0.014519 0.000434 0.004375 0.038032
0.017892 0.000326 5.73E-05 0.007137
0.005058 0.001621 0.005609 0.023775
0.034355 0.00364 0.009833 0.000124
0.007374 0.021742 0.024857 0.003578
0.009832 0.029157 0.000541 0.006228
0.032043 0.008758 9.28E-05 7.55E-05
0.018855 0.001933 0.004212 0.000681
0.009388 0.002171 5.68E-07
4.06E-06 0.000363

16.17926 @2 and 133 df

F ratio = Treatment (between group)/residual within group

 0.5 where is the variability, between or within groups?

ng for 0.95 (p = 0.05), but is our F significant??

the session send your excel file to your
have the missing 34%

ent. I'm measuring calcium inside podocytes (renal cells responsible for filtration). We have three

(AS; they cannot make one of the many collagens that hold tissue structures together)

een immortalized with a virus (HIP).

ith a dye (FURA2-AM) that is sensitive to the presence of intracellular calcium (Ca2+ ). The
background) show intracellular calcium levels after the addition of extracellular calcium. To
d each value by the respective vehicle control values. So the numbers you see are MINIMUM
calcium addition!

intracellular calcium, high fluorescence numbers indicate high intracellular calcium. The
tracellular calcium and measured intracellular calcium (the idea is that the calcium sensing
tracellular calcium in response to an elevation of extracellular calcium). Curiously before
MC, it drops!!

f podocytes (AS, NHMC, HIP) all see a similar decrease in intracellular calcium??

e are your choices; t-test, one way ANOVA, some kind of non-parametric test.

E OF TEST.............
we're taking the SSQ/df from the total pool of data points (called the total
uare, MSQ)) and subtracting away the combined variability (SSQ/df) within
each group (called the residual MSQ).

re left with is the variability BETWEEN groups (ie. treatment variability). We

pare our variability with a predicted variability from an F-distribution (it looks
a Gaussian, but is skewed to the right). If our between group variability is
than the "predicted" distribution says is expected (from random sampling),
conclude that such a big difference is UNLIKELY to occur by chance (so we're
rejecting the null hypothesis).
 Jimmy the HDR candidate wants to run an experiment to see whether a new compound ("X", synthesized by
his HDR colleague, Claire), helps restore the ability of drosophila "treated" with "mitoinhib" (a water
insoluble mitochondrial oxidative phosphorylation chain inhibitor) to climb and or grip a pole. He's
modelling Parkinson's disease (sort of).

"X" is also water insoluble (get used to it, biologists). LUCKILY both mitoinhib and "X" are soluble in 1% DMSO.

He has a total of 30 flys, he has a ruler (with a mm scale) and a stopwatch. Think about what is possible to
measure and design some sort of experiment for Jimmy. Most importantly, suggest an approach to the
analysis of the data.

Q What can he measure? How can he do it?

(1) ???? ????

(2) ???? ????

Q I've identified (maximum of) five groups for analysis. What are they?? Remember we have two c
Measurement (1)
(1) 39.6
(2) 39
(3) 12.3
(4) 62
(5) 37.9

Q Which of these groups are we most interested in comparing?

????

Q If we create a mm/s measure, is it valid??

????

Q What stats can we go with?

????

If you want 100% tell me

(1) What is the F value

(2) Is it significant? You'll need to hit the internet to find critical
(3) Draw a conclusion from this data
 d ("X", synthesized by
nhib" (a water
a pole. He's

are soluble in 1% DMSO.

t what is possible to
approach to the

member we have two compounds under investigation: mitoinhib and X!!

Measurement (1) Measurement (2)
31.2 35.7 32.2 40.1 33.3 45 52 51 30
33 34.4 31.9 39.9 36.6 50 48 39 44
18.9 10.1 16.6 19.1 6.5 23 24 26 29
70 58 51 56 54.9 52 56 59 54
33.6 34.6 30.3 38.7 35.5 46 50 47 38

internet to find critical values at whatever degrees of freedom you have

29 37
46 34
30 17
60 66
35 31