Long transcripts minus touchdown qPCR (LTMT-qPCR): a simplified and convenient method for the screening and quantification of microRNA profiles

The authors found that size selection magnetic beads could remove long RNA transcripts from total RNA with simplistic operation. After long transcript removal, microRNA could be concentrated and efficiently reverse-transcribed by stem-loop-6N primer. Touchdown qPCR improved microRNA quantification with lower C-T values and better detection efficiency. Finally, they incorporated these observations and created a new protocol named long transcripts minus touchdown qPCR for screening and quantifying microRNAs. Due to the short length and differences in abundance of microRNAs, microRNA profile screening and quantification is challenging. In this study, we found that size selection magnetic beads could be employed to easily and efficiently remove long RNA transcripts. After removing the long transcripts, the remaining small RNAs could be concentrated and then reverse-transcribed using universal stem-loop primers (USLP), with six randomized nucleotides at the 3 ' end region. The efficiency of reverse transcription decreased when the number of randomized nucleotides was reduced. In addition, we found that touchdown qPCR improved microRNA profile detection, with lower C-T values and better detection efficiency than the regular qPCR protocol, especially for those low-abundance microRNAs. Finally, we incorporated these observations to create a new protocol we named long transcripts minus touchdown qPCR (LTMT-qPCR). We performed a side-by-side comparison of LTMT with USLP and traditional stem-loop primer (TSLP) protocols. We found that LTMT has higher detection efficiency than USLP, especially for the detection of low-abundance microRNAs. Although LTMT was equivalent to TSLP in terms of microRNA profile detection, LTMT is more convenient, user-friendly, and cost-effective. Taken together, the present data indicate that LTMT is a simple, rapid, and user-friendly approach that has higher precision, accuracy, and sensitivity than the previously described methods, making it more suitable for microRNA profile screening and quantification.

Automated summary

The study demonstrates that size selection magnetic beads can efficiently remove long RNA transcripts and concentrate small RNAs for reverse transcription, while touchdown qPCR improves microRNA detection efficiency, particularly for low-abundance microRNAs. The new protocol LTMT-qPCR, integrating these techniques, shows higher precision and detection efficiency compared to traditional methods, making it more suitable for microRNA screening and quantification.