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Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay.

Zhou Y, Zhou P, Xin Y, Wang J, Zhu Z, Hu J, Wei S, Ma H - Sci Rep (2014)

Bottom Line: Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres.The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR.Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, P. R. China.

ABSTRACT
Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

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Related in: MedlinePlus

Oligonucleotides Primers Immobilization and Verification.(A) Three steps immobilization of oligonucleotides primers on polymer-coated QCM chips. Step 1: immobilization of biotin modified primers, step 2: immobilization of avidin-HRP, step 3: chemiluminescence. (B) Two steps immobilization of oligonucleotides primers on polymer-coated QCM chips. Step 1: immobilization of amido modified primer, step 2: immobilization of SYBR Green II. (C) Coloration results for (A) and (B).
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f2: Oligonucleotides Primers Immobilization and Verification.(A) Three steps immobilization of oligonucleotides primers on polymer-coated QCM chips. Step 1: immobilization of biotin modified primers, step 2: immobilization of avidin-HRP, step 3: chemiluminescence. (B) Two steps immobilization of oligonucleotides primers on polymer-coated QCM chips. Step 1: immobilization of amido modified primer, step 2: immobilization of SYBR Green II. (C) Coloration results for (A) and (B).

Mentions: Biotin-avidin detection system was applied to ensure the immobilization of oligonucleotides primer on polymer-coated QCM chips, as schematically shown in Fig. 2A. The first step was the binding of 3′ biotin modified primer on the polymer surface by amide linkage, consistent with the immobilization of Primer (1)/(2) as indicated in Methods section. The second step was the binding of avidin-HRP to the biotin modified primer. With the coverage of Luminol, the final images were presented, as shown in Fig. 2C for chemiluminescence. Evidently, image with the binding of biotin modified primer (left) owned high rate of luminous intensity, compared with the control group (right), which confirmed the immobilization of oligonucleotides primer (Fig. 2C). SYBR Green II stain was used as another approach to detect single-stranded primer immobilized on polymer-coated QCM chips (Fig. 2B). Amido modified primers were first immobilized on the polymer surface and SYBR Green II then bind to the primers and fluoresces. The results showed the significant differences between chips with primers (left panel, Fig. 2C) and chips without primers (right panel, Fig. 2C). Thus, the effectiveness of the immobilization strategy was confirmed.


Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay.

Zhou Y, Zhou P, Xin Y, Wang J, Zhu Z, Hu J, Wei S, Ma H - Sci Rep (2014)

Oligonucleotides Primers Immobilization and Verification.(A) Three steps immobilization of oligonucleotides primers on polymer-coated QCM chips. Step 1: immobilization of biotin modified primers, step 2: immobilization of avidin-HRP, step 3: chemiluminescence. (B) Two steps immobilization of oligonucleotides primers on polymer-coated QCM chips. Step 1: immobilization of amido modified primer, step 2: immobilization of SYBR Green II. (C) Coloration results for (A) and (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225532&req=5

f2: Oligonucleotides Primers Immobilization and Verification.(A) Three steps immobilization of oligonucleotides primers on polymer-coated QCM chips. Step 1: immobilization of biotin modified primers, step 2: immobilization of avidin-HRP, step 3: chemiluminescence. (B) Two steps immobilization of oligonucleotides primers on polymer-coated QCM chips. Step 1: immobilization of amido modified primer, step 2: immobilization of SYBR Green II. (C) Coloration results for (A) and (B).
Mentions: Biotin-avidin detection system was applied to ensure the immobilization of oligonucleotides primer on polymer-coated QCM chips, as schematically shown in Fig. 2A. The first step was the binding of 3′ biotin modified primer on the polymer surface by amide linkage, consistent with the immobilization of Primer (1)/(2) as indicated in Methods section. The second step was the binding of avidin-HRP to the biotin modified primer. With the coverage of Luminol, the final images were presented, as shown in Fig. 2C for chemiluminescence. Evidently, image with the binding of biotin modified primer (left) owned high rate of luminous intensity, compared with the control group (right), which confirmed the immobilization of oligonucleotides primer (Fig. 2C). SYBR Green II stain was used as another approach to detect single-stranded primer immobilized on polymer-coated QCM chips (Fig. 2B). Amido modified primers were first immobilized on the polymer surface and SYBR Green II then bind to the primers and fluoresces. The results showed the significant differences between chips with primers (left panel, Fig. 2C) and chips without primers (right panel, Fig. 2C). Thus, the effectiveness of the immobilization strategy was confirmed.

Bottom Line: Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres.The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR.Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, P. R. China.

ABSTRACT
Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

Show MeSH
Related in: MedlinePlus