RT-qPCR with chimeric dU stem-loop primer is efficient for the detection of bacterial small RNAs.
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.
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, China.
Yangfan
Y
Wu
School of Pharmacy, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, China.
Xuejiao
X
Xing
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, China.
Ting
T
You
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, China. icelike@sjtu.edu.cn.
Rubing
R
Liang
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, China. jianhualiudl@sjtu.edu.cn.
Jianhua
J
Liu
D017931:DNA Primers / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D055029:Inverted Repeat Sequences; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D058727:RNA, Small Untranslated / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D003856:Deoxyuracil Nucleotides; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D058727:RNA, Small Untranslated; D051981:Uracil-DNA Glycosidase
Deoxyuracil (dU) base; RT-qPCR; Small RNA; Stem-loop primers; Uracil-DNA glycosylase (UDG)