Volume 35, Issue 4 (July 2024)
Studies in Medical Sciences 2024, 35(4): 274-286 |
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Herischi A, Ghobeh M, Soleymani J. COMPARATIVE STUDY ON THE EFFECTS OF SEVERAL ERYTHROCYTE LYSING BUFFERS ON THE EXTRACTION OF GENOMIC DNA FROM FROZEN BLOOD SAMPLES IN EVALUATION OF TELOMERE LENGTH BY QPCR METHOD. Studies in Medical Sciences 2024; 35 (4) :274-286
URL: http://umj.umsu.ac.ir/article-1-6250-en.html
URL: http://umj.umsu.ac.ir/article-1-6250-en.html
Assistant Professor of Pharmaceutical Biomaterials, Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. , jsoleymanii@gmail.com
Abstract: (1296 Views)
Background & Aim: Studying telomere length is very important for understanding the aging process and disease prevention. The aim of this study was to study the effects of several erythrocyte lysing buffers on the extraction of genomic DNA from frozen blood samples in evaluation of telomere length by qPCR method.
Materials & Methods: In this experimental study, six different erythrocyte lysis buffers along with CTAB extraction buffer were used for DNA extraction. The quantity and purity of the extracted DNA were assessed through absorbance measurements at A260/280 and A260/230 ratios. DNA integrity was evaluated using agarose gel electrophoresis. The consistency of buffer performance was analyzed using the coefficient of variance (CV%) for DNA quantity. Also, the efficiency of PCR was reported by checking the cycle threshold (Ct) in qPCR for each sample using the linear regression equation and coefficient of determination (R2) in GraphPad Prism v10 software. Differences were considered significant at the p>0.05 level.
Results: The lysis buffer with the combination of 10 mM Tris-HCl pH 7.6 and 10 mM KCl resulted in the extraction of the highest amount of DNA (361.01 ng/µL). The results of the gel did not show any fragmentation of DNA in the samples. Absorption ratios did not show much difference between buffers. DNA extracted using a lysis buffer containing 155 mM NH4Cl, 10 mM KHCO3, 5 mM EDTA showed lower variance in DNA quantity (CV = 7.6%) and better PCR efficiency (Efficiency = 103%, R2 = 99.7%) for 36B4 and (Efficiency = 99%, R2 = 99.6%) for telomeres compared to other buffers.
Conclusion: The lysis buffer containing 155 mM NH4Cl, 10 mM KHCO3, and 5 mM EDTA had the most effective performance in extracting DNA from frozen blood samples for telomere study. This buffer with its optimal performance can be used in trials after the final result by further studies.
Materials & Methods: In this experimental study, six different erythrocyte lysis buffers along with CTAB extraction buffer were used for DNA extraction. The quantity and purity of the extracted DNA were assessed through absorbance measurements at A260/280 and A260/230 ratios. DNA integrity was evaluated using agarose gel electrophoresis. The consistency of buffer performance was analyzed using the coefficient of variance (CV%) for DNA quantity. Also, the efficiency of PCR was reported by checking the cycle threshold (Ct) in qPCR for each sample using the linear regression equation and coefficient of determination (R2) in GraphPad Prism v10 software. Differences were considered significant at the p>0.05 level.
Results: The lysis buffer with the combination of 10 mM Tris-HCl pH 7.6 and 10 mM KCl resulted in the extraction of the highest amount of DNA (361.01 ng/µL). The results of the gel did not show any fragmentation of DNA in the samples. Absorption ratios did not show much difference between buffers. DNA extracted using a lysis buffer containing 155 mM NH4Cl, 10 mM KHCO3, 5 mM EDTA showed lower variance in DNA quantity (CV = 7.6%) and better PCR efficiency (Efficiency = 103%, R2 = 99.7%) for 36B4 and (Efficiency = 99%, R2 = 99.6%) for telomeres compared to other buffers.
Conclusion: The lysis buffer containing 155 mM NH4Cl, 10 mM KHCO3, and 5 mM EDTA had the most effective performance in extracting DNA from frozen blood samples for telomere study. This buffer with its optimal performance can be used in trials after the final result by further studies.
Keywords: DNA Extraction, Frozen Blood, PCR Efficiency, Red Blood Cell Lysing Buffer, Telomere Length
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