Lab Report: Measurement of telomere length of bucall and Hek293 DNA through qPCR

Introduction

Telomeres are long repetitive sections of DNA that cap the ends of chromosomes for protection, and
to inhibit chromosomal fusion. With each replication of DNA, telomere length is shortened due to an
inability for an Okasaki fragment to be formed. The shortening of telomeres has been associated
with a number of age-associated diseases (Opresko and Shay, 2017). The enzyme telomerase
counters this shortening, and can actively increase telomere length. It works as an RNA-dependant
DNA polymerase; producing double stranded DNA by using an RNA template (Blackburn, 2004). In a
normal cell telomere shortening through replication will eventually lead to the activation of DDR,
which will halt proliferation and induce an apoptotic response through p53 mediated pathway
(Artandi and Attardi, 2005) (Reinhardt and Schumacher, 2012). This prevents the accumulation of
potentially harmful mutations; therefore somatic cells have a very low level of active telomerase.
Fetal tissue, adult germ cells, and tumour cells express active telomerase preventing this apoptotic
response.

As well as age-related diseases, active telomerase has shown a relationship with cancer. The
activation of telomerase has been shown to be an aspect of 85% of cancers (Akincilar, Unal and
Tergaonkar, 2016). Tert (telomerase reverse transcriptase), a subunit of telomerase, is involved in
this reactivation of telomerase. The Tert promoter region is subject to mutations which lead to
reactivation. This reactivation of telomerase allows cancer cells to avoid apoptosis and promote cell
proliferation. This leads to the study of telomerase being a important aspect of the efforts in cancer
research.

Human embryonic kidney 293 (HEK-293) cells are a cell line derived from an embryonic kidney in
1973. An adenoviral genome fragment of 4kbp in length was inserted into chromosome 19. This
section codes for E1A and E1B proteins that counteracts apoptosis through disruption of cell cycle
pathways (Lin et al., 2014). Being fetal tissue they express a higher level of telomerase than somatic
cells and are often used as positive controls for active telomerase, and represent standards whereby
comparable measures can be made(Fehrer et al., 2006).

This study is looking at the comparison of telomere length in buccal and Hek293 cells. As previously
discussed Hek293 cells exhibit active telomerase, thus we are expecting to see a lower Cq value
indicating a higher initial telomere length within these cells compared to the somatic buccal cells.

Methods

Six samples of buccal cells were obtained from participating individuals. Each participant used 10ml
of 0.9% saline solution to rinse their mouths for 20 seconds before collecting the buccal cell
containing solution in individual 50ml Falcon tubes. Along with these samples, twelve Human
Embryonic Kidney 293 (HEK-293) cell lines were obtained from an outside source for DNA extraction.
Centrifugation was performed on these samples at 2000 rpm for 10 minutes using a bench top
centrifuge. The aqueous layer was then removed leaving the pellet. The samples were resuspended
in 200µl of TE buffer, and vortexed to mix the solutions. 200µl of phenol/chloroform/isoamyl alcohol
was added to each of the tubes, and mixed on a vortex for 1 minute each. This mixture was then
centrifuged at 14,000 rpm for five minutes allowing the two phases to separate with the heavier
phenol solution moving towards the bottom of the tube and the aqueous/DNA phase remaining on
top. A pipette was used to remove the top aqueous layer and place it into a new tube (Tube 2), this
was ̴180µl per sample. A repeat extraction was performed on Tube one by adding 200µl of TE
buffer, followed by vortexing for 1 minute and centrifuging at 14,000 rpm for 5 minutes. Again the
aqueous layer was removed from Tube 1 and placed into Tube 2. The extracted phase in Tube 2 was
then measured and an equal volume of choloform/isoamyl was added. This mixture was then
vortexed for 1 minute and centrifuged at 14,000 rpm for 5 minutes. The aqueous phase was
carefully removed and placed in Tube 3, leaving the chloro/isoamylalcohol phase in Tube 2. A 1:10
dilution was performed on all of the Tube 3s with 3M NaOAc. Each of the tubes was measured and
2x ice cold 100% ethanol was added. These were then incubated on ice for 15 minutes. These were
then centrifuged at 14,000 rpm for 15 minutes forming a DNA pellet. The supernatant was removed,
and 300ml of 80% ice cold ethanol added. The samples were then centrifuged for 5 minutes at
14,000 rpm, before again removing the supernatant. These were air dried for 5 minutes, before
being resuspended in 200µl TE buffer and vortexed.

The samples were loaded onto the nanodrop to measure DNA concentration. The samples were
then diluted with nuclease free water to acquire samples at 10ng/µl. 2µl of each DNA sample was
loaded in duplicate into the qPCR plate as well as an NTC in duplicate. A telomere amplification
master mix was made by pipetting 50µl iQ SYBR Green Superix, 7.56µl TelG primer, 7.56µl TelC
primer, and 8.85µl nuclease-free dH2O into a tube. 10.5µl of this reaction mix was then added to
each of the DNA samples in the qPCR plate. A further 2µl of each DNA sample was then loaded in
duplicate to the empty wells of the same qPCR plate. A single copy gene amplification mix was then
made by pipetting 50µl iQ SYBR Green Superix, 5.88µl hbgD primer, 7.56µl hbdU primer, and 8.85µl
nuclease-free dH2O into a tube. 10.5 µl of this second reaction mix was added to the second set of
DNA samples on the qPCR plate. This plate was briefly spun to ensure each mixture was at the
bottom of each well.

The plate layout was as follows:

B= buccal cell DNA

H= Hek293 cell DNA

NTC= non template control

T= Telomere primers

S= Single copy gene primers

These samples were then loaded into the qPCR machine and programmed as follows:

Stage 1: 15 minutes at 95°c

Stage 2: 2 cycles of 15 seconds at 94°c; 15 seconds at 49°c

Stage 3: 27 cycles of 15 seconds at 94°c, 10 seconds at 62°c, 15 seconds at 74°c with signal
acquisition

Melt curve: 30 seconds at 95°c, 30 seconds at 65°c, 65°c to 95°c with 0.5°c increments of 30 seconds
with signal acquisition.

Results:

A NanoDrop was used to measure the DNA concentrations of the different DNA samples, as shown
below.

Tab 1. Concentration of DNA per sample analysed by NanoDrop

The qPCR produced a melt curve and amplification plots to show the temperature at which the
double stranded DNA was breaking, and the number of cycles at which each sample passed the
florescence threshold respectively.
Fig 1. Melt curve of qPCR reaction

Fig 2. Amplification plots of qPCR reaction

Data analysis was performed to show the mean Cq values for the duplicate DNA samples. Standard
deviation and coefficient of variation was also calculated to indicate the deviation from the mean of
each of the samples.
Tab2. Results table from qPCR reaction

Tab 3. Table showing Cq means across plate setup

The average coefficients for the duplicates and plate were analysed to show the variation away from
the mean of the duplicates compared to across the whole plate.

Tab 4. Table showing coefficient variants for duplicates and across plate
 Telomere Single copy Cell type Status
primer wells gene wells
A1E1 A2E2
B1F1 B2F2
C1G1 C2G2
A3E3 A4E4 Buccal Comparison possible
B3F3 B4F4
C3G3 C4G4 Hek293 Comparison possible
A5E5 A6E6
B5F5 B6F6
C5G5 C6G6
A7B7 A8B8
C7D7 C8D8
A9D9 A10D10 Buccal Comparison possible
B9E9 B10E10
C9F9 C10F10
A11D11 A12D12
B11E11 B12E12
C11F11 C12F12
Tab 5. Table showing telomere primer wells with their corresponding single gene copy primer wells. Highlighted plate positions shows
acquired data

The Telomere Cq to Single copy gene Cq ratios were calculated to show the difference in initial
product across cell types.

Tab 6. Table showing the comparable wells Cq values and telomere primers to single gene copy primers ratio

Tab 7. Table showing the average T/S ratios for Hek293 cells, bucall cells, and a ratio between them

Discussion

When analysing the qPCR data, validity of the sample must first be assured. A NanoDrop was used to
measure the concentration of DNA for each sample (Tab 1). Concentration of 10ng/µl was desired,
so samples were diluted with distilled water to meet this requirement. Samples with concentrations
lower than 10ng/µl were left undiluted but remained to be analysed through qPCR. A melt curve was
obtained from the qPCR reaction (fig.1). This showed two distinct peaks, and centralised peak at
around 77°c and another peak around 89°c. These most likely represent the telomere and single
gene copy samples respectively. The reason for the higher temperature required for the single gene
copy to break was due to the primers containing a CG clamp to increase the melting point of the
DNA. This allows us to show the amplification of both products. However, a small peak was also
observed at 68°potentially showing nonspecific amplification, or primer dimerisation. Amplification
plots were also obtained (fig 2.). This showed samples passing the florescence threshold between
the 17th and 26th cycle. This is where we would expect to see the amplification passing the threshold.
Performing this qPCR in duplicate allowed for averages to be calculated and thus produced more
reliable measurements for Cq values (Tab 3.). Where data was only observed from one of the
duplicates the other was discarded and the singular well data was analysed. Using duplicated also
allowed for standard deviations to be calculated, to express how far from the mean each sample
was. These appeared to be relatively low (0.3-2.9), indicating conformity of the acquired data. This
was also backed up by the coefficient of variation of the duplicates being low. We compared the CoV
of the duplicates to that of the entire plate (Tab 4.). This allows us to show that difference between
the duplicates is less than that of the plate, 6.2% and 10.6% respectively.

Each DNA sample was treated with single copy gene primers and telomere primers. This would
allow us to show a direct comparison between the lengths of telomeres in buccal cells compared to
Hek293 cells. However, the qPCR did not produce data for ever well which makes comparisons
difficult. As shown in Table 5, the highlighted wells indicate a result for the qPCR with a Cq value
obtained. For a comparison to be possible a DNA sample must have both a Cq value for the telomere
primers and single copy gene primers as this allows for a ratio to be calculated. As indicated in table
5 there were only 3 DNA samples that had a positive Cq value for both single copy gene primers and
telomere primers. These were buccal cells A3E3:E4, Hek293 cells G3:G4, and buccal cells D9:D10.

This comparable data allows us to look at when the single copy gene and telomere amplifications
pass the florescence threshold for three different DN samples. The data shows (Tab 6.) that in each
case the telomere Cq was lower than the single gene copy Cq. As with each cycle of the qPCR
doubling the amount of target DNA for each primer, a lower Cq indicates a higher initial number of
places to primers can anneal and produce florescence. This is what we expect from the repetitive
nature of telomeres.

Finally, when looking at the buccal cell DNA compared to Hek293 cell DNA, the ratio shows very little
difference (Tab 7.). This is not what we would expect as Hek293 cells have been shown to have long
telomeres and active telomerase. This cell line is sometimes used as a positive control when looking
at telomerase activity (Schmelzer and Reid, 2009). This is not a significant result however, as the
sample size is extremely small and isn’t indicative of buccal cell to Hek293 telomerase activity ratios.

Errors may have been introduced at different stages during the DNA extraction and qPCR leading to
the loss of significant data. Pipetting error, despite being small may have interfered with the
concentration of DNA in each sample. Contamination of the samples may have been introduced and,
is shown to be likely due with multiple peaks in the melt curve. I would suggest that this experiment
is performed again to attempt to obtain more data, leading to a stronger statistical power.

References

Akincilar, S. C., Unal, B. and Tergaonkar, V. (2016) ‘Reactivation of telomerase in cancer’, Cellular
and Molecular Life Sciences, 73(8), pp. 1659–1670.
Artandi, S. E. and Attardi, L. D. (2005) ‘Pathways connecting telomeres and p53 in senescence,
apoptosis, and cancer’, Biochemical and Biophysical Research Communications, 331(3), pp. 881–890.

Blackburn, E. (2004) ‘Telomeres and telomerase: their mechanisms of action and the effects of
altering their functions’, FEBS Letters, 579(4), pp. 859–862.

Fehrer, C. et al. (2006) ‘Techniques in gerontology: Cell lines as standards for telomere length and
telomerase activity assessment’, Experimental Gerontology, 41(6), pp. 648–651.

Lin, Y. C. et al. (2014) ‘Genome dynamics of the human embryonic kidney 293 lineage in response to
cell biology manipulations’, Nature Communications, 5(2), p. 4767.

Opresko, P. L. and Shay, J. W. (2017) ‘Telomere-associated aging disorders’, Ageing Research

Reviews, 33(1), pp. 52–66.

Reinhardt, H. C. and Schumacher, B. (2012) ‘The p53 network: Cellular and systemic DNA damage
responses in aging and cancer’, Trends in Genetics, 28(3), pp. 128–136.

Schmelzer, E. and Reid, L. M. (2009) ‘Human telomerase activity, telomerase and telomeric template
expression in hepatic stem cells and in livers from fetal and postnatal donors’, European Journal of
Gastroenterology and Hepatology, 21(10), pp. 1191–1198.