TKTL1 expression in human malign and benign cell lines

BMC Cancer

Table 5 Comparison of PCR efficiency for the TKTL1 gene obtained with the three primer pairs

Cell line	Primer pairs	E (%)	Slope	R²	Cq (1:5)	Cq (1:320)	Cq (NTC)
HEK293-TKTL1	TKTL1(1)	----	-----	----	35.38 ± 0.78	35.95 ± 0.58	>40
	TKTL1(2)	95.7	−3.429	0.991	22.09 ± 0.04	28.29 ± 0.46	>38
	TKTL1(3)	94.9	−3.451	0.997	22.05 ± 0.24	28.14 ± 0.20	>40
	PPIA	94.2	−3.470	0.994	22.78 ± 0.25	29.15 ± 0.10	>38
	β-actin	95.1	−3.445	0.999	19.63 ± 0.03	25.89 ± 0.02	>32
HEK293-control	TKTL1(1)	-----	-----	-----	33.74 ± 1.19	35.89 ± 0.85	>37
	TKTL1(2)	-----	-----	-----	27.78 ± 0.03	27.98 ± 0.02	>37
	TKTL1(3)	-----	-----	-----	30.33 ± 0.51	33.87 ± 1.01	>35
	PPIA	90.2	−3.581	0.999	17.39 ± 0.05	23.94 ± 0.08	>35
	β-actin	91.1	−3.554	1.000	17.38 ± 0.08	23.79 ± 0.06	>31
U251	TKTL1(1)	100.3	−3.315	0.987	26.44 ± 0.32	32.49 ± 0.10	>39
	TKTL1(2)	103.5	−3.242	0.997	27.78 ± 0.02	33.69 ± 0.42	>36
	TKTL1(3)	103.3	−3.246	0.989	26.69 ± 0.10	32.88 ± 0.04	>36
	PPIA	99.3	−3.339	0.996	18.92 ± 0.07	25.08 ± 0.07	>37
	β-actin	99.3	−3.339	1.000	15.53 ± 0.02	21.57 ± 0.02	>31
JAR	TKTL1(1)	94.3	−3.467	0.987	27.67 ± 0.567	33.93 ± 0.421	>40
	TKTL1(2)	92.1	−3528	0.974	28.64 ± 0.26	35.42 ± 0.53	>36
	TKTL1(3)	90.1	−3.584	0.938	27.91 ± 064	34.48 ± 0.56	>37
	PPIA	93.5	−3.489	0.995	14.82 ± 0.01	24.99 ± 0.05	>40
	β-actin	98.1	−3.367	0.999	14.82 ± 0.01	20.96 ± 0.02	>31

The results are shown for the HEK293-TKTL1 and HEK293-control transfectants as well as for the cell lines JAR and U251 with the highest expression levels. The following information about the performance of RT-qPCR for the three TKTL1 primer pairs as well as for the primer pairs for reference (housekeeping) genes β-actin and PPIA is given: the PCR efficiency (E in %) and the slope of the standard curves (a PCR efficiency of 100% corresponds to a slope of −3.32 and a slope of less −3.32 is indicative of a PCR efficiency <100%), the correlation coefficient R² reflecting the linearity of the standard curve (ideally, R² = 0.999) and the quantification (threshold) cycles Cq for cDNA dilutions 1:5 and 1:320. In addition, Cq values for no template controls (NTC) are shown. The amplification efficiencies of TKTL1 gene and the endogenous references genes β-actin and PPIA are comparable. It is worth to mention that the performance of PCR with the three primer pairs did not demonstrate a dynamic range for the enzyme reaction allowing to construct a standard curve (copy number versus Cq value), and therefore to calculate the efficiency of the enzyme reaction.

ISSN: 1471-2407