NDLI: RT-qPCR with chimeric dU stem-loop primer is efficient for the detection of bacterial small RNAs

Content Provider	Springer Nature Link
Author	Wu, Yangfan Xing, Xuejiao You, Ting Liang, Rubing Liu, Jianhua
Copyright Year	2017
Abstract	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.
Starting Page	4561
Ending Page	4568
Page Count	8
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	101
Issue Number	11
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2017-03-17
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Small RNA RT-qPCR Stem-loop primers Deoxyuracil (dU) base Uracil-DNA glycosylase (UDG) Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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