尽管microRNA芯片和microRNA测序检测方法已经普遍使用,但qRT-PCR依旧是检验microRNA表达定量的金标准。 由于microRNA的结构特殊,长度只有18-25个碱基,无法直接采用常规的PCR技术扩增,因此RT-qPCR的引物设计对很多刚刚接触microRNA的同学都是一个难题。在针对microRNA的PCR技术中,设计其引物的理念是基于延长待测microRNA的长度,构建出一个足够长的PCR模板,才能进一步应用PCR技术来定量分析。 最常用的microRNA 反转录PCR方法就是茎环法(stem-loop)和加尾法(poly-A tail)。由于茎环法反转录的引物设计原理限制,加尾法的检测通量比茎环法更高,因此加尾法也被实验室普遍使用。 今天小编就给大家介绍一个批量设计microRNA加尾法反转录PCR引物的软件miRprimer,重点是一键完成哦! miRprimer 官方推荐下载网站:https:///projects/miRprimer/ 网站后台文件直接下载miRprimer地址: https://jaist.dl./project/miRprimer/miRprimer2_installer.zip miRprimer2_installer.zip,整个软件的压缩包只有2.68M (2848kb)大小。 提醒: 1. 软件支持在Windows XP或更高系统中运行,还没在苹果电脑Mac OS系统测试(原因是小编的钱包羞涩~~) 2. 经小编测试,最新版本miRprimer顺利运行,不需在电脑中安装Ruby脚本环境。 第一步 miRprimer2_installer.zip压缩包2.68M大小,下载后解压缩生成同名文件夹,内有三个文件:input_miRs.txt、miRprimer2.exe、README.txt。 特别提示:不要更改这些文件的文件名。 第二步 input_miRs.txt,fasta格式储存的是需要设计引物的microRNA名字和序列。 可以按照模板形式输入待设计引物的microRNA名字和序列: >miR name microRNA sequence 第三步 双击exe程序之后,会出现一个DOS界面。这时引物设计就已经完成了(意不意外,惊不惊喜?~~)。这时文件夹会出现5个新txt文件,随手点击键盘enter后,DOS界面消失。 ——恭喜你,引物设计完成了! 小编推荐用MS Excel程序打开查看使用,方便编辑复制提交。 result_best_primer_pairs.txt文件包含了每个microRNA最佳引物的评分、正向和反向引物的评分,以及引物二聚体形成的评分。这里只有评分最高的引物对,小编推荐优先采纳这个文件中给出的最佳引物对,将序列提交引物合成服务商。 如果上面提供的最佳引物实验效果不佳,或者溶解曲线不理想的话,其他四个文件就派上用场。 result_all_primer_pairs.txt文件包含了每个microRNA的所有引物对评分和引物二聚体的评分信息。 result_f_primers.txt和result_r_primers.txt文件,包含了每个正向引物和反向引物的长度、评分,基于评分的排名和详细评分数;
result_comparison_of_pairs.txt文件,目的是便于人工检查所有正向和反向引物间两两之间序列相似度差异。Similarty to other pairs相似度越低,引物间差异越大;序列完全相同的引物,相似度是1。 如果软件提供的最佳引物对在反转录中工作不如意,不如试试那些相似度低的备选引物试试看。 那么问题来了,有朋友问小编: 人和其他模式动物 [1] miR-181d regulates human dendritic cell maturation through NF-κB pathway. Cell Prolif. 2017 Jul 21. doi: 10.1111/cpr.12358. (人microRNA) [2] MicroRNA profiling of low-grade glial and glioneuronal tumors shows an independent role for cluster 14q32.31 member miR-487b. Mod Pathol. 2017 Feb;30(2):204-216. doi: 10.1038/modpathol.2016.177. (人microRNA) [3] Leptin and insulin up-regulate miR-4443 to suppress NCOA1 and TRAF4, and decrease the invasiveness of human colon cancer cells. BMC Cancer. 2016 Nov 14;16(1):882. (人microRNA) [4] An miRNA-mediated therapy for SCA6 blocks IRES-driven translation of the CACNA1A second cistron. Sci Transl Med. 2016 Jul 13;8(347):347ra94. doi: 10.1126/scitranslmed.aaf5660. (人microRNA) [5] Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner. Mol Neurobiol. 2017 Aug;54(6):4584-4596. doi: 10.1007/s12035-016-9974-3. (小鼠microRNA) [6] Circulating miRNAs in Murine Experimental Endometriosis. Reprod Sci. 2017 Mar;24(3):376-381. doi: 10.1177/1933719116667228. (小鼠microRNA) [7] Dissecting genetics of cutaneous miRNA in a mouse model of an autoimmune blistering disease. BMC Genomics. 2016 Feb 16;17:112. doi: 10.1186/s12864-016-2455-2. (小鼠microRNA) [8] Rapamycin (Sirolimus) alters mechanistic target of rapamycin pathway regulation and microRNA expression in mouse meiotic spermatocytes. Andrology. 2015 Sep;3(5):979-90. doi: 10.1111/andr.12075. (小鼠microRNA) [9] Impact of 17beta-estradiol and progesterone on inflammatory and apoptotic microRNA expression after ischemia in a rat model. J Steroid Biochem Mol Biol. 2017 Mar;167:126-134. doi: 10.1016/j.jsbmb.2016.11.018. (大鼠microRNA) [10] Circulating let-7g is down-regulated in Bernese Mountain dogs with disseminated histiocytic sarcoma and carcinomas - a prospective study. Vet Comp Oncol. 2017 Jun;15(2):525-533. doi: 10.1111/vco.12196. (狗microRNA) [11] Joint Profiling of miRNAs and mRNAs Reveals miRNA Mediated Gene Regulation in the Göttingen Minipig Obesity Model. PLoS One. 2016 Nov 30;11(11):e0167285. doi: 10.1371/journal.pone.0167285. (小型猪microRNA) [12] Hepatic expression of inflammatory genes and microRNAs in pigs with high 'cholesteryl ester transfer protein' (CETP) activity. Mamm Genome. 2016 Oct;27(9-10):503-10. doi: 10.1007/s00335-016-9649-4. (猪microRNA) [13] Transcriptional analysis of porcine intestinal mucosa infected with Salmonella Typhimurium revealed a massive inflammatory response and disruption of bile acid absorption in ileum. Vet Res. 2016 Jan 7;47:11. doi: 10.1186/s13567-015-0286-9. (猪microRNA) [14] Gender and Obesity Specific MicroRNA Expression in Adipose Tissue from Lean and Obese Pigs. PLoS One. 2015 Jul 29;10(7):e0131650. doi: 10.1371/journal.pone.0131650. (猪microRNA)
植物 [15] Time-Course Small RNA Profiling Reveals Rice miRNAs and Their Target Genes in Response to Rice Stripe Virus Infection. PLoS One. 2016 Sep 14;11(9):e0162319. doi: 10.1371/journal.pone.0162319. (水稻microRNA) [16] Small RNA sequencing and degradome analysis of developing fibers of short fiber mutants Ligon-lintles-1 (Li 1 ) and -2 (Li 2 ) revealed a role for miRNAs and their targets in cotton fiber elongation. BMC Genomics. 2016 May 17;17:360. doi: 10.1186/s12864-016-2715-1. (棉花microRNA) [17] Identification of novel small ncRNAs in pollen of tomato. BMC Genomics. 2015 Sep 18;16:714. doi: 10.1186/s12864-015-1901-x. (西红柿small non-coding RNA)
微生物 [18] Manipulation of Viral MicroRNAs as a Potential Antiviral Strategy for the Treatment of Cytomegalovirus Infection. Viruses. 2017 May 19;9(5). (病毒microRNA) [19] Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia. Genes (Basel). 2016 Dec 20;7(12). pii: E131. doi: 10.3390/genes7120131. (鞭毛虫microRNA) [20] EBV and not HPV sensitizes tobacco-associated head and neck cancer cell line FaDu to radiotherapy. Acta Otolaryngol. 2016;136(4):354-62. doi: 10.3109/00016489.2015.1114182. (病毒和人microRNA)
综述和方法学文献 [21] Considerations for optimization of microRNA PCR assays for molecular diagnosis. Expert Rev Mol Diagn. 2016;16(4):407-14. doi: 10.1586/14737159.2016.1152184. (综述) [22] Towards the identification of the loci of adaptive evolution. Methods Ecol Evol. 2015 Apr;6(4):445-464. (综述) [23] MicroRNAome genome: a treasure for cancer diagnosis and therapy. CA Cancer J Clin. 2014 Sep-Oct;64(5):311-36. doi: 10.3322/caac.21244. (综述) [24] Quantification of miRNAs by a simple and specific qPCR method. Methods Mol Biol. 2014;1182:73-81. doi: 10.1007/978-1-4939-1062-5_7. (方法学) |
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