The MiraMas™ Kit is designed to create cDNA libraries for qPCR detection of microRNAs and other small RNAs. The method is based on ligation of a 5’- adenylated/3’ blocked oligonucleotide adapter (Adenylated 3’ Ligation Adapter) to the 3’ ends of the small RNAs using RNA Ligase, followed by reverse transcription with M-MLV Reverse Transcriptase to convert the small RNAs to cDNA templates for qPCR. The ligated adapter provides the binding site for the reverse transcription (RT) primer. After the second-strand cDNA is synthesized by extension of a microRNA-specific Forward PCR primer, the same adapter sequence serves as binding site for the Reverse PCR primer. Use of an adenylated adapter is advantageous because it streamlines the work-flow and leads to increased specificity for producing the desired reaction products, with fewer unwanted side products. The MiraMas protocol uses a single-tube format for ligation, reverse transcription, and subsequent dilution of the cDNA library. The MiraMas Kit allows users great flexibility in formatting their experiments, in that the number of samples that can be processed is scaled to the amount of sample RNA used in the reactions. Depending on the amount of sample RNA, the kit can be used to produce cDNA libraries from 30 to 150 samples. Each cDNA library can be used for detection of tens to hundreds of target microRNAs. The kit includes enough Universal Reverse Primer for 1,000 real-time PCRs.
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