fa بهینه‌سازی استخراج DNA برای مطالعه طول تلومر از نمونه‌های خون منجمد بیوشیمی Biochemistry. Cell Biology and Genetics پژوهشي Original <div style="text-align: justify;"><span style="font-size:12pt"><span style="line-height:115%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#1f4e79">زمینه:</span></span></span></span></b><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> بهینه‌سازی استخراج </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">DNA</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> در راستای حفظ طول تلومر به‌عنوان زیست‌نشانگر پیری و انواع بیماری‌ها بسیار مهم است. نمونه‌های خونی که به‌طور طولانی مدت نگهداری شده‌اند، منبع بسیار مهمی برای مطالعات ژنتیکی محسوب می‌شوند. در این مطالعه با استفاده از بافرهای لیز مختلف به همراه بافر استخراج </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">CTAB</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">، کیفیت </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">DNA</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> استخراجی و اثر بازدارنده‌های همراه آن بر روی </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">PCR</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> کمی در مطالعات تلومر بررسی شد.</span></span></span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="line-height:115%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#1f4e79">مواد و روش‌ها:</span></span></span></span></b><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> استخراج </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">DNA</span></span></span></span><span style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> با ۶ بافر لیزکننده گلبول قرمز و بافر استخراج بر پایه </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">CTAB</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">، انجام شد. یکپارچگی </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">DNA</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> با ژل الکتروفورز، کمّیت با جذب در ۲۶۰ نانومتر و خلوص آن با مقادیر مورد انتظار ۸/۱ و ۲/۲-۲ برای نسبت‌های </span></span></span></span>_{<span lang="FA" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۲۸۰</span></span></span></span>}<span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">A</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">/</span></span></span></span>_{<span lang="FA" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۲۶۰</span></span></span></span>}<span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">A</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> و _۲۳۰</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">A</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">/</span></span></span></span>_{<span lang="FA" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۲۶۰</span></span></span></span>}<span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">A</span></span></span></span><span style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> ارزیابی گردید. ارزیابی کمّی اثر هر بافر در واکنش </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">q-PCR</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> و تکرارپذیری نتایج به ترتیب با محاسبه بازده واکنش، ضریب تعیین (</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">R²</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">) و درصد ضریب تغییرات (</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">%CV</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">)</span></span></span></span> <span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">محاسبه شد.</span></span></span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="line-height:115%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#1f4e79">یافته‌ها:</span></span></span></span></b><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> بیش‌ترین مقدار </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">DNA</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> استخراج شده (324/93 نانوگرم بر میلی‌لیتر، 11/53</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">CV%:</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">) با استفاده از بافر ۵ (۱۰ میلی‌مولار </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">Tris-HCL </span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۶/۷</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">pH=</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">، ۵۰ میلی‌مولار </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">NaCl</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">) به‌دست آمد. تمامی بافرها مقادیر مطلوب برای </span></span></span></span>_{<span lang="FA" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۲۸۰</span></span></span></span>}<span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">A</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">/</span></span></span></span>_{<span lang="FA" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۲۶۰</span></span></span></span>}<span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">A</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> و </span></span></span></span>_{<span lang="FA" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۲۳۰</span></span></span></span>}<span dir="LTR" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">A</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">/</span></span></span></span>_{<span lang="FA" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۲۶۰</span></span></span></span>}<span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">A</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> داشتند و از نظر خلوص </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">DNA</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> تفاوتی (0/05</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">p></span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">) نداشتند. طبق نتایج ژل، </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">DNA</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> استخراج شده همه بافرها به جز یکی، فاقد خردشدگی بود. مولکول </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">DNA</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> استخراج شده با بافر ۱ (</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">NH</span></span></span></span>_{<span dir="LTR" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">4</span></span></span></span>}<span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">Cl </span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۱۵۵میلی‌مولار، </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">KHCO</span></span></span></span>_{<span dir="LTR" style="font-size:11.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">3</span></span></span></span>} <span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۱۰ میلی‌مولار و </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">EDTA </span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">۵ میلی‌مولار) بهترین عملکرد را در واکنش </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">q-PCR</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> برای ژن </span></span></span></span><i><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">HBG</span></span></span></span></i><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> (0/126=</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">E</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> و 0/97=</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">R²</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">) و تلومر (0/99= بازده، 0/99=</span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">R²</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">) داشت.</span></span></span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="line-height:115%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#1f4e79">نتیجه‌گیری:</span></span></span></span></b><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> مولکول </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">DNA</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> استخراج شده با بافر ۱ از نمونه خون منجمد جهت مطالعه تلومر کم‌ترین بازدارندگی </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d">q-PCR</span></span></span></span><span lang="FA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"> را نشان داد.</span></span></span></span><span lang="AR-SA" style="font-size:10.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#0d0d0d"></span></span></span></span></span></span></span></span></span></div> <div style="text-align: justify;"><span style="font-size:12pt"><span style="line-height:115%"><span new="" roman="" style="font-family:" times=""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#002060">Background:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#404040">Optimizing DNA extraction for the preservation of telomere length &ndash; as a biomarker for ageing and vari-ous diseases &ndash; is highly important. Long-term stored blood samples are considered essential resources for genetic studies. In this study, different lysis buffers were used along with CTAB extraction buffer to inves-tigate the quality of extracted DNA and the effect of its accompanying inhibitors on quantitative PCR (q-PCR) in telomere studies.</span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="line-height:115%"><span new="" roman="" style="font-family:" times=""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#002060">Materials and Methods:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#404040">DNA extraction was performed using six RBC lysis buffers and CTAB-based extraction buffer. DNA integrity was evaluated by gel electrophoresis, quantity by absorbance at 260 nm and its purity with expected values of 1.8 and 2-2.2 for the ratios of A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀. The quantitative effect of each buffer in q-PCR and the repeatability of the results were assessed by calculating the reaction efficiency, coefficient of determination (R²), and percentage of coefficient of variation (%CV).</span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="line-height:115%"><span new="" roman="" style="font-family:" times=""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#002060">Results:</span></span></span></span></b> <span style="font-size:9.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#404040">Buffer number 5 (10 mM Tris-HCl, pH 7.6, 50 mM NaCl) yielded the highest amount of DNA extraction (324.93 ng/ml, %CV 11.53). All the extracted DNA samples were pure, as indicated by the acceptable A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ratios (p>0.05). Gel analysis revealed that the extracted DNA of all the buffers ex-cept one was intact. The DNA molecule extracted with buffer number 1 (155 mM NH4Cl, 10 mM KHCO₃, and 5 mM EDTA) showed the best performance in q-PCR for <i>HBG</i> gene (efficiency=0.126, R²=0.97) and telomere (efficiency=0.99, R²=0.99).</span></span></span></span></span></span></span><br> <span style="font-size:12pt"><span style="line-height:115%"><span new="" roman="" style="font-family:" times=""><b><span style="font-size:9.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#002060">Conclusion: </span></span></span></span></b><span style="font-size:9.0pt"><span style="line-height:115%"><span iransans="" style="font-family:"><span style="color:#404040">The DNA molecule extracted from frozen blood samples by buffer number 1 showed the least q-PCR inhibition for telomere study.</span></span></span></span></span></span></span></div> تلومر, استخراج, هموگلوبین, خون, بافر Telomere, DNA extraction, Hemoglobin, Blood, Buffer 37 52 http://ismj.bpums.ac.ir/browse.php?a_code=A-10-1115-12&slc_lang=fa&sid=1 Sajedeh Sobhanparast ساجده سبحان‌پرست 5700319475328460016204 0009-0008-3311-7893 No Department of Biology, School of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran <br>Tuberculosis & Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran گروه زیست‌شناسی، دانشکده علوم پایه، دانشگاه شهید مدنی آذربایجان، تبریز، ایران<br>مرکز تحقیقات سل و بیماری‌های ریه، دانشگاه علوم پزشکی تبریز، تبریز، ایران Jafar Soleymani جعفر سلیمانی 5700319475328460016205 5700319475328460016205 No Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran مرکز تحقیقات آنالیز دارویی، دانشگاه علوم پزشکی تبریز، تبریز، ایران Younes Aftabi یونس آفتابی 5700319475328460016206 5700319475328460016206 No Tuberculosis & Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran مرکز تحقیقات سل و بیماری‌های ریه، دانشگاه علوم پزشکی تبریز، تبریز، ایران Nader Chaparzadeh نادر چاپارزاده Nchapar@azaruniv.ac.ir 5700319475328460016207 5700319475328460016207 Yes Department of Biology, School of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran گروه زیست‌شناسی، دانشکده علوم پایه، دانشگاه شهید مدنی آذربایجان، تبریز، ایران