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Comparative quantification of plasma TDRD7 mRNA in cataract patients by real-time polymerase chain reaction.

Kim ST, Chun JW, Park G, Koh JW - Korean J Ophthalmol (2014)

Bottom Line: After polymerase chain reaction, the results were compared after quantifying the TDRD7 mRNA using ABL1 mRNA for normalization.We analyzed the relative gene expression data via the ΔΔCt method.The comparison of the genetic values of different types of cataracts demonstrated that the TDRD7 expression level of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017).

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Chosun University College of Medicine, Gwangju, Korea.

ABSTRACT

Purpose: To investigate the relationship between plasma TDRD7 mRNA and lens transparency, and to evaluate plasma TDRD7 mRNA as a potential marker for cataracts and its sub-type by quantitatively analyzing human peripheral blood.

Methods: Plasma RNA was extracted from 40 patients with cataracts, and 30 normal controls of matched age and gender. Blood cholesterol and fasting glucose were measured, and the RNA extracted from the sample was synthesized into cDNA. After polymerase chain reaction, the results were compared after quantifying the TDRD7 mRNA using ABL1 mRNA for normalization. We analyzed the relative gene expression data via the ΔΔCt method.

Results: The normalized 2(-ΔΔCt) of plasma TDRD7 mRNA based on ABL1 mRNA was 1.52 ± 0.63 in the case of the control group and 1.05 ± 0.34 in the case of the cataract patients, and the TDRD7 expression level of the cataract patients was lower than that of the control group (p = 0.048). The comparison of the genetic values of different types of cataracts demonstrated that the TDRD7 expression level of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017).

Conclusions: Human cataracts and the TDRD7 gene loss-of-function mutations are strongly causally related, as the expression level of plasma TDRD7 mRNA in patients with cataracts was statistically significantly lower than in the normal control group.

No MeSH data available.


Related in: MedlinePlus

Comparison of gene expression of ABL1 and TDRD7 genes in cataract group versus normal group by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Amplification curves for A) ABL1, B) TDRD7 (normal group), and C) TDRD7 (cataract group). The threshold level is given by a horizontal line. The cycle at which the mean amplification curve of ABL1 or TDRD7 real-time RT-PCRs crosses the threshold (cycle threshold value) is indicated by a vertical line. RFU = relative fluorescence units.
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Figure 1: Comparison of gene expression of ABL1 and TDRD7 genes in cataract group versus normal group by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Amplification curves for A) ABL1, B) TDRD7 (normal group), and C) TDRD7 (cataract group). The threshold level is given by a horizontal line. The cycle at which the mean amplification curve of ABL1 or TDRD7 real-time RT-PCRs crosses the threshold (cycle threshold value) is indicated by a vertical line. RFU = relative fluorescence units.

Mentions: Total RNA was extracted from each venous blood sample with the EasyRed RNA extraction kit (Intronbio, Seoul, Korea) in accordance with the manufacturer's protocol. The cDNA synthesis was conducted using a Superscript VILO cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol. For quantification of the TDRD7 transcript relative to the ABL1 control gene, we used the primers specific for TDRD7 (forward: 5'-ACGAGTAGAGATCACAAATG-3'; reverse: 5'-TCTGCTAAACAGCACTTAAT-3') and ABL1 (forward 5'-CTGCTAGAGAAGGACTACC-3'; reverse: 5'-TCGTCTGAGATACTGGATTC-3'). Real-time polymerase chain reaction (PCR) reactions with the above primers were conducted in a total volume of 20 µL using Phusion Flash (Thermo Scientific, Wilmington, DE, USA) with EvaGreen (Biotium, Hayward, CA, USA) in accordance with the manufacturer's recommendations. Thermocycling was carried out using a CFX96 real-time PCR detection system (Bio-rad, Hercules, CA, USA) with the following conditions: 1 cycle of 98℃ for 10 seconds, 40 cycles of 98℃ for 1 second, and 55℃ for 15 seconds. The amplification curves of 2 genes, TDRD7 and ABL1 mRNA, are shown in Fig. 1.


Comparative quantification of plasma TDRD7 mRNA in cataract patients by real-time polymerase chain reaction.

Kim ST, Chun JW, Park G, Koh JW - Korean J Ophthalmol (2014)

Comparison of gene expression of ABL1 and TDRD7 genes in cataract group versus normal group by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Amplification curves for A) ABL1, B) TDRD7 (normal group), and C) TDRD7 (cataract group). The threshold level is given by a horizontal line. The cycle at which the mean amplification curve of ABL1 or TDRD7 real-time RT-PCRs crosses the threshold (cycle threshold value) is indicated by a vertical line. RFU = relative fluorescence units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120356&req=5

Figure 1: Comparison of gene expression of ABL1 and TDRD7 genes in cataract group versus normal group by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Amplification curves for A) ABL1, B) TDRD7 (normal group), and C) TDRD7 (cataract group). The threshold level is given by a horizontal line. The cycle at which the mean amplification curve of ABL1 or TDRD7 real-time RT-PCRs crosses the threshold (cycle threshold value) is indicated by a vertical line. RFU = relative fluorescence units.
Mentions: Total RNA was extracted from each venous blood sample with the EasyRed RNA extraction kit (Intronbio, Seoul, Korea) in accordance with the manufacturer's protocol. The cDNA synthesis was conducted using a Superscript VILO cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol. For quantification of the TDRD7 transcript relative to the ABL1 control gene, we used the primers specific for TDRD7 (forward: 5'-ACGAGTAGAGATCACAAATG-3'; reverse: 5'-TCTGCTAAACAGCACTTAAT-3') and ABL1 (forward 5'-CTGCTAGAGAAGGACTACC-3'; reverse: 5'-TCGTCTGAGATACTGGATTC-3'). Real-time polymerase chain reaction (PCR) reactions with the above primers were conducted in a total volume of 20 µL using Phusion Flash (Thermo Scientific, Wilmington, DE, USA) with EvaGreen (Biotium, Hayward, CA, USA) in accordance with the manufacturer's recommendations. Thermocycling was carried out using a CFX96 real-time PCR detection system (Bio-rad, Hercules, CA, USA) with the following conditions: 1 cycle of 98℃ for 10 seconds, 40 cycles of 98℃ for 1 second, and 55℃ for 15 seconds. The amplification curves of 2 genes, TDRD7 and ABL1 mRNA, are shown in Fig. 1.

Bottom Line: After polymerase chain reaction, the results were compared after quantifying the TDRD7 mRNA using ABL1 mRNA for normalization.We analyzed the relative gene expression data via the ΔΔCt method.The comparison of the genetic values of different types of cataracts demonstrated that the TDRD7 expression level of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017).

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Chosun University College of Medicine, Gwangju, Korea.

ABSTRACT

Purpose: To investigate the relationship between plasma TDRD7 mRNA and lens transparency, and to evaluate plasma TDRD7 mRNA as a potential marker for cataracts and its sub-type by quantitatively analyzing human peripheral blood.

Methods: Plasma RNA was extracted from 40 patients with cataracts, and 30 normal controls of matched age and gender. Blood cholesterol and fasting glucose were measured, and the RNA extracted from the sample was synthesized into cDNA. After polymerase chain reaction, the results were compared after quantifying the TDRD7 mRNA using ABL1 mRNA for normalization. We analyzed the relative gene expression data via the ΔΔCt method.

Results: The normalized 2(-ΔΔCt) of plasma TDRD7 mRNA based on ABL1 mRNA was 1.52 ± 0.63 in the case of the control group and 1.05 ± 0.34 in the case of the cataract patients, and the TDRD7 expression level of the cataract patients was lower than that of the control group (p = 0.048). The comparison of the genetic values of different types of cataracts demonstrated that the TDRD7 expression level of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017).

Conclusions: Human cataracts and the TDRD7 gene loss-of-function mutations are strongly causally related, as the expression level of plasma TDRD7 mRNA in patients with cataracts was statistically significantly lower than in the normal control group.

No MeSH data available.


Related in: MedlinePlus