Copy Number Analysis for CHO cells via TaqMan

Design of Primers and Probe

Design probe over an intron/exon boundary of the hamster sequence if possible. Use Primer
Express Software to find the best primer/probe pairs. The probe should contain more C’s than
G’s, reverse the strand if necessary.

Resuspend primers at 300uM with TE buffer

Resuspend probe at 100uM with TE buffer

Titration of primers and probe:

Calculate the standard curve to test the primer and probe concentration

Calculate the mass of a single plasmid molecule

m = mass
n = plasmid size m = [n] [1.096e-21 g/bp]
e-21 = x10-21

Calculate the mass of the plasmid containing the copy#’s of interest that is 3,000,000 to 30
copies.

Example:
pGB 20 is 6900bp
One copy weighs 7.5624e-18 g

[g] needed per ul for 5ul

copies plasmid [g] input
3000000 2.27E-11 4.54E-12
300000 2.27E-12 4.54E-13
30000 2.27E-13 4.54E-14
3000 2.27E-14 4.54E-15
300 2.27E-15 4.54E-16
30 2.27E-16 4.54E-17
3 2.27E-17 4.54E-18
1 7.5624E-18 1.51E-18

Quantitate the plasmid containing the sequence to be amplified and dilute the standard curve
based on the calculations above (dilute the primers on the bench designated to plasmids only).
Vortex before each dilution.
 plasmid plasmid dilution final
g/ul stock x1 x100 diluentx1 x100 factor concentration
pGB20 undiluted 172.7 ng/ul
1:10 1.73E-07 undiluted 1 10 9 90 10 1.73E-08
1:100 1.73E-08 1:10 1 10 9 90 10 1.73E-09
1:1,000 1.73E-09 1:100 1 10 9 90 10 1.73E-10
3e6 std 1.73E-10 1:1000 1 30.00 37.06 1111.8 38.06 4.54E-12
3e5 std 4.54E-12 3e6 std 1 100 9 900 10 4.54E-13
3e4 std 4.54E-13 3e5 std 1 100 9 900 10 4.54E-14
3e3 std 4.54E-14 3e4 std 1 100 9 900 10 4.54E-15
3e2 std 4.54E-15 3e3 std 1 100 9 900 10 4.54E-16

Do single test run to test primers and probe first

It is necessary to pipette the different elements of the assay in different locations to avoid
contamination.

In the lab upstairs:

Dilute primers to 4uM from the Master Stock (dilution 1:75) → 2.6 ul in 200ul
Dilute probe to 2uM from the Master Stock (dilution 1:50) → 4ul in 200ul

Aliquot TE buffer for the “No-Template-Control” (NTC)

In the hood:
The genomic DNA is diluted to 10ng/ul before the assay.

Make up master mix for all of the reactions.

For each TaqMan reaction use 45ul of master mix and 5 ul of template, the standards are
pipetted in triplicate and the unknowns are pipetted 8 times. For each set of samples a cocktail
is made up and then pipetted out into the multiple wells.
180 ul master mix + 20ul 3e6 standard (x4) pipet in triplicate
180 ul master mix + 20ul 3e5 standard (x4) pipet in triplicate
180 ul master mix + 20ul 3e4 standard (x4) pipet in triplicate
180 ul master mix + 20ul 3e3 standard (x4) pipet in triplicate
180 ul master mix + 20ul 3e2 standard (x4) pipet in triplicate

405 ul master mix + 45 ul DXB11 New (x9) pipet into 8 wells

405 ul master mix + 45 ul TE New (x9) pipet into 8 wells

Master Mix 1 reaction

Reaction Component Volume for one Final Concentration
reaction
TaqMan Master Mix 25 ul 1x
forward primer [4uM] 2.5 ul 200 nM
reverse primer [4 uM] 2.5 ul 200 nM
probe [2uM] 2.5 ul 100 nM
DNA sample 5 ul 1ng/ ul
H2O 12.5 ul -
38 reactions, make enough for 40 reactions:
Reaction Component Volume for 40 Final Concentration
reaction
TaqMan Master Mix 1000 ul 1x
forward primer [4uM] 100 ul 200 nM
reverse primer [4 uM] 100 ul 200 nM
probe [2uM] 100 ul 100 nM
DNA sample - -
H2O 500 ul -

Vortex and pipette 50 ul of each cocktail very carefully into TaqMan plates and cap the plate
with optical caps.

Pipette the NTC first and cap the column.

Pipette the CHO control and cap the column.
Pipette any DNA samples and cap the columns.

On the bench:
Pipette the plasmid standards starting with the lowest concentration and ending with the
highest. Cap the remaining wells and be sure the caps are on securely.
Run on the machine using the standard protocol.

1 2 3 4 5 6 7 8 9 10 11 12
A 3e6 3e6 3e6 CHO NTC

B 3e5 3e5 3e5 CHO NTC

C 3e4 3e4 3e4 CHO NTC

D 3e3 3e3 3e3 CHO NTC

E 3e2 3e2 3e2 CHO NTC

F CHO NTC

G CHO NTC

H Test plate CHO NTC

After it is confirmed that the primers and probe are functional, the primers need to be titrated
against the probe. The purpose of this procedure is to determine the minimum primer
concentrations that give the maximum ΔRn with the lowest threshold cycle (CT).
Use the following matrix to set up the titration:

Forward Primer [nM]

Reverse primer
[nM] 50 300 900

50 50/50 300/50 900/50

300 50/300 300/300 900/300
900 50/900 300/900 900/900

Make up enough of each concentration to be able to do all conditions in quadruplicate:

A B C
final final final
Component conc. 5rxn Component conc. 5rxn Component conc. 5rxn
PCR mix 1x 125.00 PCR mix 1x 125.00 PCR mix 1x 125.00
For primer 50 nM 3.13 For primer 50 nM 3.13 For primer 50 nM 3.13
Rev primer 50 nM 3.13 Rev primer 300 nM 18.75 Rev primer 900 nM 56.25
Probe 250 nM 31.25 Probe 250 nM 31.25 Probe 250 nM 31.25
H20 87.50 H20 71.88 H20 34.38
template 5ul 25.00 template 5ul 25.00 template 5ul 25.00
final volume 50ul 250 final volume 45ul 250 final volume 45ul 250

D E F
final final final
Component conc. 5rxn Component conc. 5rxn Component conc. 5rxn
PCR mix 1x 125.00 PCR mix 1x 125 PCR mix 1x 125
For primer 300 nM 18.75 For primer 300 nM 18.75 For primer 300 nM 18.75
Rev primer 50 nM 3.13 Rev primer 300 nM 18.75 Rev primer 900 nM 56.25
Probe 250 nM 31.25 Probe 250 nM 31.25 Probe 250 nM 31.25
H20 71.88 H20 56.25 H20 18.75
template 5ul 25.00 template 5ul 25 template 5ul 25
final volume 50ul 250 final volume 45ul 250 final volume 45ul 250

G H I
final final final
Component conc. 5rxn Component conc. 5rxn Component conc. 5rxn
PCR mix 1x 125.00 PCR mix 1x 125 PCR mix 1x 125
For primer 900 nM 56.25 For primer 900 nM 56.25 For primer 900 nM 56.25
Rev primer 50 nM 3.13 Rev primer 300 nM 18.75 Rev primer 900 nM 56.25
Probe 250 nM 31.25 Probe 250 nM 31.25 Probe 250 nM 31.25
H20 34.38 H20 18.75 H20 -18.75
template 5ul 25 template 5ul 25 template 5ul 25
final volume 50ul 250 final volume 45ul 250 final volume 45ul 250

Add 25 ul of the 3e3 standard point into each tube and load 50 ul per well:
 1 2 3 4 5 6 7 8 9 10 11 12
A (A) (B) (C)
50/50 50/50 50/50 50/300 50/300 50/300 50/900 50/900 50/900

B (D) (E) (F)

300/50 300/50 300/50 300/300 300/300 300/300 300/900 300/900 300/900

C (G) (H) (I)

900/50 900/50 900/50 900/300 900/300 900/300 900/900 900/900 900/900

H Titration plate

Tabulate the ΔRn values and CT values for each condition and pick the primer probe pair that
has the highest ΔRn and lowest CT value. The probe is kept at 250 nM and does not need to be
titrated for this application.
After the primer/ probe concentration are established the reagents need to be aliquoted into
single-use aliquots and frozen at -20°C. Label tubes with primer name, concentration, date and
initials

Qualification of the Assay

Before the genomic DNA samples can be run, the test range and reproducibility of the assay
need to be determined. This is done by running the standard curve with the conditions
established in the titration experiment above several times. A second set of standard will be run
in the same plate as an “unknown” and the quantity of the standard measured. Afterwards the
standards will be examined for reproducibility and for their limit of quantitation (LOQ)

Make up separate cocktails for the standards and the standards being run as unknowns.

1 2 3 4 5 6 7 8 9 10 11 12
A 3e6 3e6 3e6 3e6 3e6 3e6 CHO NTC

B 3e5 3e5 3e5 3e5 3e5 3e5 CHO NTC

C 3e4 3e4 3e4 3e4 3e4 3e4 CHO NTC

D 3e3 3e3 3e3 3e3 3e3 3e3 CHO NTC

E 3e2 3e2 3e2 3e2 3e2 3e2 CHO NTC

F CHO NTC

G CHO NTC

H LOQ plate CHO NTC

Parameters that should be met by the assay:

1) Calculate the % CV for the Ct values and the sample quantity values

%CV= (stdev/ average)*100

%CV of Ct values for all samples should be less than ~1.0%

%CV of Qty values for samples should be less than ~ 10%

2) Calculate the LOQ for the standards that were run as unknowns

%LOQ= (measured qty/ expected qty)*100

The LOQ should be between 80-120%

The unknown samples for the genomic DNA need to fall in the range of the standard
curve that meet this criteria

3) Parameters for the standard curve

%CV of Ct values for all samples should be less than ~1.0%
r2 value should be > 0.99
slope should be < -3.3
Three plates minimum need to be run for the LOQ experiment. All plates will be
analyzed using the same threshold and baseline. All standard curves can then be
compared between experiments. CV’s of standards from plate to plate also need to be
calculated and shouldn’t exceed 2%.