Wu Yangfan, Xing Xuejiao, You Ting, Liang Rubing, Liu Jianhua
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, China.
School of Pharmacy, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, China.
Appl Microbiol Biotechnol. 2017 Jun;101(11):4561-4568. doi: 10.1007/s00253-017-8181-0. Epub 2017 Mar 17.
Small non-coding RNAs are considered be involved in the regulation of multiple cellular processes. Quantitative reverse transcription PCR (RT-qPCR) is widely used in the detection of eukaryotic microRNA, and the stem-loop primers can improve the specificity and efficiency of reverse transcription. However, the loop structure of primers probably influence the next quantitative amplification due to the base stacking and steric hindrance. Here, we designed a chimeric stem-loop primer with a deoxyuracil (dU) base located near the RNA matching part. After the reverse transcription, uracil-DNA glycosylase (UDG) treatment was used to remove the dU base and destroy the stem-loop structure of RT product. Enzymatic assay confirmed that the recombinant UDG could efficiently eliminate the dU base in the oligonucleotide. Transcriptions of two small RNAs (TFF and ryeA) in Escherichia coli were detected by RT-qPCR with different primers. Results showed that the use of the chimeric dU stem-loop primer and UDG treatment could enhance the detection specificity and sensitivity about 1.1- to 3.4-fold, compared to those with traditional stem-loop primer and linear primer. Total RNA of 1-10 pg was enough for efficient detection with the chimeric stem-loop primers. In a word, this strategy could promote the RT-qPCR detection efficiency on the transcription of bacterial small RNAs even in trace samples and can facilitate the detection of exiguous change in cellular metabolism.
小非编码RNA被认为参与多种细胞过程的调控。定量逆转录PCR（RT-qPCR）广泛用于真核微小RNA的检测，茎环引物可提高逆转录的特异性和效率。然而，由于碱基堆积和空间位阻，引物的环结构可能会影响下一步的定量扩增。在此，我们设计了一种嵌合茎环引物，其脱氧尿嘧啶（dU）碱基位于RNA匹配部分附近。逆转录后，使用尿嘧啶-DNA糖基化酶（UDG）处理去除dU碱基并破坏RT产物的茎环结构。酶活性测定证实重组UDG可有效消除寡核苷酸中的dU碱基。使用不同引物通过RT-qPCR检测大肠杆菌中两种小RNA（TFF和ryeA）的转录。结果表明，与传统茎环引物和线性引物相比，使用嵌合dU茎环引物和UDG处理可将检测特异性和灵敏度提高约1.1至3.4倍。1-10 pg的总RNA足以使用嵌合茎环引物进行有效检测。总之，该策略可提高RT-qPCR对细菌小RNA转录的检测效率，即使在微量样品中也是如此，并有助于检测细胞代谢中的微小变化。
