!

Efficacy'of'GAPDH'
and'ALAS2'as'
Reference'Genes'
Connecticut)College)
Deborah)Eastman,)Brad)DeMarco)16,)
Abbas)Zaidi)16,)Julie)Noet)16)

ABSTRACT'
Testing(the(effectiveness(of(
GAPDH(and(ALAS2(as(reference(
genes(in(pharengeal(endoderm(
cDNA(of(Ambystoma(Mexicanum(
through(RTCqPCR.()
!

2'
!

EFFICACY'OF'GAPDH'AND'ALAS2'AS'REFERENCE'GENES)

Introduction
The Notch signaling pathway is a
highly
conserved
juxtacrine
signaling
mechanism between cells. This pathway plays a
key role in the cell determination in both
vertebrate and invertebrate cells. The Notch
pathway also is highly involved in regulation of
apoptosis and cell proliferation within
organisms.
The
Notch
protein
is
a
transmembrane protein that regulates the
production of transcription factors that alter gene
expression.
Many degenerative and cancerous
conditions are related to the abnormal
functionality of the Notch protein. Ambystoma
Mexicanum were used as a model organism as
their taste buds are easily accessible and
differentiate from multipotent epithelial cells to
developed taste buds throughout their embryotic
stages. Taste bud development occurs within the
oropharyngeal endoderm tissue, making the
tissue easily assessable. Not much is known
about the cellular mechanisms that drive the
differentiation of taste buds from pharyngeal
epithelia. Gastrulation is finished by Ambystoma
Mexicanum at stage 15, at which the
specification of the epithelium is complete
(Figure 1). Taste bud cells will autonomously
develop into patterned taste bud progenitor cells

from the pharyngeal endoderm once gastrulation

is finished (Parker et al, 2004). Differentiation
into matured taste buds from progenitor cells
isnt completed until the 9th day of development
of Ambystoma Mexicanum (Figure 1).

Figure 1: Timeline of taste bud development of Ambystoma

Mexicanum (Parker et al, 2004)

The ability to assay for Notch genes in the

differentiation of Ambystoma Mexicanum can
help further understand the pathway and key
genes that are involved. Accurate gene assay
protocol is crucial towards finding accurate
results. Reference genes are necessary to
normalize expression patterns measured by RTqPCR without the need to equalize cDNA
concentrations. An important characteristic of
ideal reference gene in this experiment is
constant and equal expression experimental
conditions or tissue type. Furthermore,
constituent primers of an ideal reference gene
must have high efficiency in varying
concentrations of template DNA. Currently, H4
serves as a dependable reference gene with a
high efficiency. H4 codes for histone H4, a
crucial protein in the organization of DNA in
cells. This suggests the genes equal expression
throughout tissue types.

Table A: Target genes and qPCR primer pairs for candidate reference genes (Guelke et al, 2015)

This experiment investigated two new possible

reference gene candidates in Ambystoma
Mexicanum proposed by Guelke (Table A);
GAPDH and ALAS2. Traditionally, GAPDH has
been deemed an inappropriate reference gene
(Guelke et al, 2015); however, GAPDHs
reference gene viability in developing
oropharyngeal endoderm tissue was investigated.
GAPDH codes for the cytosolic enzyme
glyceraldehyde 3-phosphate dehydrogenase, a
critical enzyme in glycolysis. This makes the
prevalence of GAPDH crucial in meeting cells
energy demands, suggesting equal expression
throughout
tissue.
ALAS2
codes
for
Aminoluvulinic synthase, an important enzyme

in heme synthesis. This enzyme catalyses the

conversion of succinyl-CoA to d-aminolevulunic acid in the mitochondria of cells.
Expression of ALAS2 has been found primaryly
in bone marrow, spleen, and eryothrocytes of H.
Sapiens, M. musculus, and R. norvegicus
(BioGPS)(Figure B). Efficiencies of each of the
constituent primers for ALAS2 and GAPDH
were quantified using cDNA isolated from larval
head tissue of Ambystoma Mexicanum.
Furthermore, expression deviation throughout
developmental stages was measured in GAPDH
and compared to that of H4 to determine
viability of GAPDH as a reference gene.

EFFICACY'OF'GAPDH'AND'ALAS2'AS'REFERENCE'GENES'

3)

Figure A: Expression pattern of GAPDH in H. Sapien

(BioGPS)

Figure B: Expression pattern of ALAS2 in H. Sapiens

(BioGPS)

Our results found that GAPDH serves as a

reliable reference gene, with a primer efficiency
of 95.5% and equal or lower variability than H4.
ALAS2 showed lackluster results, with little to
no registered cycle thresholds in initial RT-qPCR
reactions.
Materials and Methods
RNA Preparation
Ambystoma Mexicanum sample was transferred
to a mortar tube with Buffer RLT (600L). The
sample was ground with a pestle, transferred to a
microcentrifuge tube, and homogenized. The
sample was then centrifuged at 10k RPM for 3
minutes and the supernatant was transferred to a
microcentrifuge tube with 70% ethanol (600L).
700L of this solution was transferred into an
RNeasyTM spin column and centrifuge at 10k
RPM for 15 seconds. The flow-through was
discarded and Buffer RWI (700L) was added
and then centrifuged at 10k RPM for 15 seconds.

The flow-through was discarded and Buffer RPE

(500L) was added and then centrifuged at 10k
RPM for 15 seconds and the flow-through was
discarded. Buffer RPE (500L) was added and
the sample was spun at 10k for 2 minutes and the
flow-through was discarded. The RNeasyTM
spin column was placed into 1.5mL tube. 40mL
of RNase-free water was added and the sample
was centrifuged at 10k RPM for 1 minute and
the flow-through was frozen.
cDNA Preparation
The prepared RNA (10L), olgio dT (1L), and
dNTP (1L) was added to a microcentrifuge
tube. The mixture was placed in a 65C water

4'
!

EFFICACY'OF'GAPDH'AND'ALAS2'AS'REFERENCE'GENES)

bath for 5 minutes, immediately chilled on ice,

then centrifuge at 10k RPM for 15 seconds. 5X
First Strand (4L), DTT (0.1M, 2L), and
RNase out (1L) were added and incubated at
42C for 2 minutes. SuperscriptTM II RT (1L)
was added and the solution was incubated at
42C for 50 minutes and 70C for 15 minutes.
qPCR Design: Primer Efficiency
Primers following Guelke were reconstituted
from lyophilization to 10M stock solutions. 1:5,
1:50, 1:500, 1:5000, and 1:25000 dilutions of
target cDNA were prepared. A mastermix was
prepared using 1L of cDNA (1:5, 1:50, 1:500,
1:5000, or 1:25000 dilution), 10L SYBR green
(Biorad), and 8L of ddH2O. 19L of mastermix
were allocated to each respective qPCR-plate
well with 1L of a constituent primer (10M).
RT-qPCR was run using a Biorad
thermocycler under user defined settings (Table
1a).
# of Cycles

Temperature
95C

3:00

39

95C

0:10

65C

0:30

65C

0:05

95C
curve)

(melt

Gel Electrophoresis
200mL of TBE solution (0.5X) was used to
prepare a 2% agarose gel. DNA ladder and
qPCR products were loaded onto the gel with
SYBR safe loading dye (Biorad) (Table 1b). The
gel was run for one hour at 100 constant volts
and an image was taken using a Geldoc.
Lane

Component

Volume
Component
(L)

Volume
loading dye
(L)

Volume
ddH2O
(L)

DNA Ladder

DNA Ladder

GADPH 1:5
PCR product

GAPDH 1:50
PCR product

GAPDH
1:500 PCR
product

GADPH
1:5000 PCR
product

GAPDH
1:25k PCR
product

H4 1:5 PCR
product

GAPDH 1:5
PCR product

10

GAPDH
1:5000 PCR
product

Time (mins)

allocated to each respective qPCR-plate well

with 1L of a constituent primer (10M). RTqPCR was run using a Biorad thermocycler
under user defined settings (Table 1a).

N/A

Table 1a: Thermocycler settings for RT-qPCR reaction

qPCR Design: Standardization of Expression

A 1:50 dilution was prepared of cDNA from
different developmental stages (LH, 15, 28, 30,
32). Absorbance was taken at 260nm to assay for
cDNA concentration. The concentrations were
then equalized via dilution. . A mastermix was
prepared using 1L of cDNA (standardized LH,
15, 28, 30, or 32), 10L SYBR green (Biorad),
and 8L of ddH2O. 19L of mastermix were

Table 1b: Lane

electrophoresis

allocation

of

components

to

gel

EFFICACY'OF'GAPDH'AND'ALAS2'AS'REFERENCE'GENES'

5)

Results
Primer

Dilution

Cq

H4

Unkn

1:05

18.69

H4

Unkn

1:05

17.88

GAPDH

Unkn

1:05

22.87

GAPDH

Unkn

1:50

29.68

GAPDH

Unkn

1:500

33.85

GAPDH

Unkn

1:5000

36.92

GAPDH

Unkn

1:25000

N/A

GAPDH

Unkn

1:05

24.02

GAPDH

Unkn

1:50

27.22

GAPDH

Unkn

1:500

31.52

GAPDH

Unkn

1:5000

36.22

GAPDH

Unkn

1:25000

35.56

GAPDH

Unkn

1:05

30.67

GAPDH

Unkn

1:50

GAPDH

Unkn

GAPDH
GAPDH

Sample

Efficiency

GAPDH Triplicate 1

63.4%

GAPDH Triplicate 2

94.6%

GAPDH Triplicate 3

N/A

Table 2b: Calculated primer efficiencies of GAPDH from

primer efficiency regression plot (Figure 1) (Appendix A).
Target

Content

Sample

Cq

H4

Unkn

1:5

20.65

H4

Unkn

1:5

19.92

GAPDH

Unkn

1:5

22.81

GAPDH

Unkn

1:50

27.21

GAPDH

Unkn

1:500

31.28

GAPDH

Unkn

1:5000

34.95

27.56

GAPDH

Unkn

1:25000

31.70

1:500

31.07

GAPDH

Unkn

1:5

23.71

Unkn

1:5000

N/A

GAPDH

Unkn

1:50

26.84

Unkn

1:25000

N/A

GAPDH

Unkn

1:500

31.61

GAPDH

Unkn

1:5000

34.12

GAPDH

Unkn

1:25000

33.16

GAPDH

Unkn

1:5

GAPDH

Unkn

1:50

31.86

GAPDH

Unkn

1:500

36.26

GAPDH

Unkn

1:5000

36.15

GAPDH

Unkn

1:25000

39.74

Table 2a: Quantified cycle threshold values for RT-qPCR

run on LH dilutions with H4 and GAPDH primers.

40!

GAPDH!
Duplicate!1!
y!=!$4.632x!+!20.644!
R!=!0.9674!

35!

GAPDH!
Duplicate!2!

30!

y!=!$3.4594x!+!21.779!
R!=!0.95377!
GAPDH!
Duplicate!3!
y!=!$0.2x!+!29.427!
R!=!0.01083!

Cq#

25!
20!

Table 3a: Quantified cycle threshold values for RT-qPCR

run on LH dilutions with H4 and GAPDH primers.

Linear!
(GAPDH!
Duplicate!1)!

15!

45.00!

Linear!
(GAPDH!
Duplicate!2)!

0!
$4!

$3!

$2!

Log([cDNA])#

$1!

0!

Linear!
(GAPDH!
Duplicate!3)!

35.00!
30.00!
25.00!

Cq#

5!

$5!

GAPDH!
Triplicate!1!
y!=!$4.0476x!+!20.16!
R!=!0.99836!
GAPDH!
Triplicate!2!
y!=!$3.6004x!+!21.151!
R!=!0.98677!
GAPDH!
Triplicate!3!
y!=!$2.554x!+!28.026!
R!=!0.8712!
Linear!(GAPDH!
Triplicate!1)!

40.00!

10!

20.00!
15.00!
10.00!

Figure 1: Primer efficiency regression run plotting Log([LH

cDNA]) versus obtained Cq (Table 2a). A linear regression
was run on each triplicate set.

Linear!(GAPDH!
Triplicate!2)!

5.00!
0.00!
$5!

$4!

$3!

$2!

$1!

0!

Linear!(GAPDH!
Triplicate!3)!

Log([cDNA])#

Figure 2: Primer efficiency regression run plotting Log([LH

cDNA]) versus obtained Cq (Table 3a). A linear regression
was run on each triplicate set.

6'
!

EFFICACY'OF'GAPDH'AND'ALAS2'AS'REFERENCE'GENES)

Sample

Efficiency

Quantifying Deviation of Gene Expression

GAPDH Triplicate 1

76.6%

Target

Content

Sample

Cq

GAPDH Triplicate 2

89.6%

GAPDH

Unkn

LH

22.95

GAPDH

Unkn

15

29.08

GAPDH Triplicate 3

146.3%

GAPDH

Unkn

28

32.30

Table 3b: Calculated primer efficiencies of GAPDH from

primer efficiency regression plot (Figure 2) (Appendix A).

GAPDH

Unkn

30

22.44

GAPDH

Unkn

32

23.46

Target

Content

Sample

Cq

GAPDH

Unkn

LH

22.60

H4

Unkn

1:5

24.54

GAPDH

Unkn

15

26.72

H4

Unkn

1:5

25.22

GAPDH

Unkn

28

25.17

ALAS2

Unkn

1:5

36.08

GAPDH

Unkn

30

21.10

ALAS2

Unkn

1:50

GAPDH

Unkn

32

21.40

ALAS2

Unkn

1:500

H4

Unkn

LH

18.17

ALAS2

Unkn

1:5000

H4

Unkn

15

22.68

ALAS2

Unkn

1:25000

H4

Unkn

28

22.39

ALAS2

Unkn

1:5

36.76

H4

Unkn

30

23.46

ALAS2

Unkn

1:50

37.75

H4

Unkn

32

23.53

ALAS2

Unkn

1:500

H4

Unkn

LH

17.08

ALAS2

Unkn

1:5000

H4

Unkn

15

23.00

ALAS2

Unkn

1:25000

H4

Unkn

28

21.23

ALAS2

Unkn

1:5

H4

Unkn

30

23.26

ALAS2

Unkn

1:50

H4

Unkn

32

37.23

ALAS2

Unkn

1:500

ALAS2

Unkn

1:5000

ALAS2

Unkn

1:25000

Table 5a: Quantified cycle threshold values from RT-qPCR

using H4 and GAPDH primers on oropharyngeal endoderm
cDNA and various stages of development of larval head
cDNA. Concentrations of cDNA were equalized using
spectrophotometry and dilution.

34.44

Table 4a: Quantified cycle threshold values for RT-qPCR

run on LH dilutions with H4 and ALAS2 primers.

RT-qPCR performed with a dilution series of

larval head cDNA yielded a primer efficiency
ranging from 76.6%-95.5% efficiency in
GAPDH. Preliminary ALAS2 efficiency
calculations were unsuccessful with little to no
registered cycle threshold values.

Gene

Standard
Deviation
Cq

Average Cq
Standard
Deviation

GAPDH Duplicate 1

4.40

3.32

GAPDH Duplicate 2

2.46

H4 Duplicate 1

2.22

H4 Duplicate 2

7.61

4.91

Table 5b: Calculated standard deviation of registered cycle

threshold values (Table 5a) of GAPDH and H4 primer
duplicates from different stages of development of
oropharyngeal endoderm tissue.

EFFICACY'OF'GAPDH'AND'ALAS2'AS'REFERENCE'GENES'

7)

!
Target

Content

Sample

Cq

GAPDH

Unkn

LH

21.51

GAPDH

Unkn

15

35.18

GAPDH

Unkn

28

36.11

GAPDH

Unkn

30

39.77

GAPDH

Unkn

32

27.46

GAPDH

Unkn

LH

21.57

GAPDH

Unkn

15

36.41

GAPDH

Unkn

28

38.84

GAPDH

Unkn

30

GAPDH

Unkn

32

29.47

H4

Unkn

LH

19.59

H4

Unkn

15

30.19

H4

Unkn

28

25.81

H4

Unkn

30

31.16

H4

Unkn

32

H4

Unkn

LH

H4

Unkn

15

H4

Unkn

28

H4

Unkn

30

H4

Unkn

32

Gene

Average Cq
Deviation

GAPDH

5.40

H4

5.09

Standard

Table 7: Average Cq standard deviation in GAPDH and H4

from both data sets (Table 6b, Table 5b).

Deviation of expression of GADPH ranged from

2.46 to 9.35. Averaged deviation from all RTqPCR revealed an average Cq deviation of 5.40.
This is relatively low when compared to H4,
which yielded a standard deviation of 5.09 in
similar experimental conditions.
Electrophoresis of RT-qPCR Products

Table 6a: Quantified cycle threshold values from RT-qPCR

using H4 and GAPDH primers on oropharyngeal endoderm
cDNA and various stages of development of larval head
cDNA. Concentrations of cDNA were equalized using
spectrophotometry and dilution.
Gene

Standard
Deviation Cq

Average Cq
Standard
Deviation

GAPDH Duplicate 1

7.38

7.49

GAPDH Duplicate 2

9.35

H4 Duplicate 1

5.27

H4 Duplicate 2

N/A

5.27

Table 6b: Calculated standard deviation of registered cycle

threshold values (Table 6a) of GAPDH and H4 primer
duplicates from different stages of development of
oropharyngeal endoderm tissue.

Figure 3: Electrophoresis of RT-qPCR products of serial

dilutions of larval head cDNA using GAPDH and H4
primers.

The electrophoresis of the qPCR products

showed that GADPH yielded a single band that
had similar size for all qPCR reactions with that
primer. qPCR on larval head using H4 primers
yielded a single band at a different size than
GAPDH. Size cannot be accurately determined
due to the lack of a DNA ladder.

8'
!

EFFICACY'OF'GAPDH'AND'ALAS2'AS'REFERENCE'GENES)

Discussion
Initial primer efficiency assay proved
that the proposed GAPDH primer is highly
efficient at replicating the GAPDH gene. Most
assays yielded an efficiency of 80% or more with
only one assay with efficiency below 80%. All
GAPDH PCR products had single bands at
relatively equivalent positions. Although the size
cannot be determined from the gel, the similar
banding patterns suggest that the same PCR
products were produced in all PCR reactions.
The presence of a single band also suggests that
no unexpected PCR products were formed as
well as primer dimers, which would interfere
with replication efficiency. Furthermore, the
deviation of GAPDH expression throughout
developing pharyngeal endoderm and larval head
tissue was relatively low. The average calculated
deviation of expression was found to be 5.40.
H4, a commonly used reference gene registered
an average deviation of 5.09, suggesting
GAPDH has a very low variability of expression
relative to H4. This observation is substantiated
due to the prevalence of GAPDH expression
throughout tissue. Glycolytic enzymes such as
glyceraldehyde 3-phosphate dehydrogenase is
essential in allowing cells to meet general energy
requirements, even in anaerobic condition and
tissue that only uses glycolysis for ATP
production (such as erythrocytes). H. sapien
expression patterns also gave inclination of
relatively equal expression of GAPDH
throughout tissue types (Figure A).
Primer efficiency calculations of
ALAS2 proved to be very unsuccessful. Most
RT-qPCR reaction resulted in unregistered cycle
threshold values even at 1:5 dilutions. Initial
registered ALAS2 cycle threshold value of most
concentrated cDNA yielded cycle threshold from
34.4-36.76 (Table 4a). This high cycle thresholds
suggests two possibilities: (1) the design of an
inefficient ALAS2 or (2) the lack of expression
of ALAS2 in larval head tissue of Ambystoma
Mexicanum. If the former is the case, further
PCR design must be performed. A possible route
to testing the causes of the lack of efficiency is a
gel electrophoresis of ALAS2 PCR products
from LH cDNA template. The later is most
likely the cause of the observed inefficiencies of
ALAS2 primers in PCR reactions. We can
explain
this
through
the
biochemical
involvement of aminoluvulinic synthase, which
is coded by ALAS2. This enzyme is critical in
porphyrin ring synthesis, catalyzing the initial

conversion of succinyl-CoA to d-aminolevulunic acid in the mitochondria. The most

prevalent porphyrin ring is biology is the heme
group, which is predominately present in
erythrocytes and erythrocyte progenitors, such as
bone marrow. This observation also shows itself
in expression patterns of H. Sapiens, where a
majority of expression is seen in early erythroid
cells and bone marrow with little to no
expression seen in other tissue (Figure B). This
later observation rules out ALAS2 as a reliable
reference gene as high variance in expression
would be expected if the proposed theory were
true.
Some systematic were seen throughout the
experiment, which had significant effect on the
earlier registered cycle threshold values. Air
bubbles present in the PCR reaction mixture had
a significant impact on the Cq values, causing
even H4 to register high or even zero initial cycle
threshold values. This problem was solved
through spinning of the PCR plates to remove
any air that could affect the reaction. Future
research will be conducted by investigating the
efficacy of other primer candidates proposed by
Guelke.
Conclusion
GAPDH proved an effective primer
with initial studies suggesting high primer
efficiency and low deviation in expression
throughout varying tissue samples. Initial studies
on ALAS2 proved unsuccessful, which little to
no
registered
Cq
values,
suggesting
inefficiencies in primer design or lack of
expression of ALAS2 in target tissues.
Acknowledgements
Julie Noet and Abbas Zaidi were an
integral part of a cohesive research team and this
could not be done without their assistance.
Thanks again to Professor Eastman for her
guidance and encouragement to strive for
meaningful results.

EFFICACY'OF'GAPDH'AND'ALAS2'AS'REFERENCE'GENES'
!

Appendix
Calculation of Primer Efficiency
! = 3.4594! + 21.779!
! = 3.4594!
!

! = (10!! 1) 100%!
!

! = (10!!!.!"#! 1) 100%!
! = 0.955 100%!
! = 95.5%

References
Bordzilovskaya, N.P., T.A. Dettlaff, Susan T.
Duhon, and George M. Malacinski. 1989.
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by J.B. Armstrong and G.M. Malacinski. Oxford
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Martin Baron, An overview of the Notch
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Developmental Biology, Volume 14, Issue 2,
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http://dx.doi.org/10.1016/S1084-9521(02)001799.
Parker A., Mark, et al. 2004. Cell ContactDependent Mechanisms Specify Taste Bud
Pattern During a Critical Period Early in
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Dehydrogenase)." BioGPS Blog. N.p., n.d. Web.
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9)