Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies
Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies
复制标题
DOI：
10.1186/1746-1596-4-9
复制
发表时间：
2009-03-24
影响因子：
2.6
通讯作者：
Fehrenbach, Heinz
中科院分区：
文献类型：
--
作者：
Muyal, Jai Prakash;Muyal, Vandana;Fehrenbach, Heinz
背景资料：基于基因表达谱的技术检测代表原始样品的转录差异的可靠性受到提取的RNA质量的影响。这完全取决于所使用的技术。因此，本研究旨在系统地比较硅胶柱（SGC）和异硫氰酸胍（GTC）技术的RNA分离，以回答以下问题：当冷冻、长期储存或新鲜肺组织必须进行下游分子分析时，哪种技术更可取。通过在-80 ℃长期储存（2.5年）制备冷冻肺（n = 3），同时收获新鲜肺（n = 3）并立即处理。用分光光度计测定RNA的纯度和定量，用iCycler检测系统测定靶序列的总拷贝数，以评估RNA的完整性（28 S和18 S）和片段大小，即短结果：1）在mRNA基序的3'端分别有3个长度分别为200 bp、700 bp和1400 bp的非翻译区（GAPDH-3' UTR）、中等长度（GAPDH）和长长度（PBGD）的序列。在冷冻和新鲜组织中，GTC的RNA总产量高于SGC技术，而RNA纯度保持相当。定量逆转录-聚合酶链反应数据显示，较高的平均拷贝数的28 S和一个较长的片段（1400 bp），从分离的RNA与SGC比GTC技术使用新鲜以及冷冻组织。此外，仅在用SGC技术从新鲜组织中分离的RNA中获得了高平均拷贝数的18 S和中等片段（700 bp）。对于较短的片段，这两种技术之间没有显着差异noticed.Conclusion：我们的数据表明，虽然GTC技术产生了较高的RNA量，SGC技术是更优越的上级方面的可靠生成的完整的RNA和有效地扩增较长的产品在新鲜以及在冷冻组织。
Background: The reliability of gene expression profiling-based technologies to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. It strictly depends upon the technique that has been employed. Hence, the present study aimed at systematically comparing silica-gel column (SGC) and guanidine isothiocyanate (GTC) techniques of RNA isolation to answer the question which technique is preferable when frozen, long-term stored or fresh lung tissues have to be evaluated for the downstream molecular analysis.Methods: Frozen lungs (n = 3) were prepared by long-term storage (2.5 yrs) in -80 C while fresh lungs (n = 3) were harvested and processed immediately. The purity and quantification of RNA was determined with a spectrophotometer whereas the total amounted copy numbers of target sequences were determined with iCycler detection system for assessment of RNA intactness (28S and 18S) and fragment sizes, i.e. short (GAPDH-3' UTR), medium (GAPDH), and long (PBGD) with 200 bp, 700 bp, and 1400 bp distance to the 3' ends of mRNA motif, respectively.Results: Total yield of RNA was higher with GTC than SGC technique in frozen as well as fresh tissues while the purity of RNA remained comparable. The quantitative reverse transcriptase-polymerase chain reaction data revealed that higher mean copy numbers of 28S and a longer fragment (1400 bp) were obtained from RNA isolated with SGC than GTC technique using fresh as well as frozen tissues. Additionally, a high mean copy number of 18S and medium fragment (700 bp) were obtained in RNA isolated with SGC technique from fresh tissues, only. For the shorter fragment, no significant differences between both techniques were noticed.Conclusion: Our data demonstrated that although the GTC technique has yielded a higher amount of RNA, the SGC technique was much more superior with respect to the reliable generation of an intact RNA and effectively amplified longer products in fresh as well as in frozen tissues.