Mir-X™ miRNA qRT-PCR SYBR^® Kits are complete, dual-function systems for performing first-strand cDNA synthesis and quantitative PCR (qPCR) to precisely measure the level of your favorite miRNAs. The kits are available in economical, large-sized formats that provide 200 or 600 qPCR reactions, and each kit includes a Mir-X miRNA First-Strand Synthesis Kit and SYBR^® Advantage^® qPCR Premix.  

Simple and Sensitive

A simple, single-step reaction uses an optimized mix of poly(A) polymerase and SMART™ MMLV Reverse Transcriptase to synthesize first-strand cDNA from your RNA sample (Figure 1). The cDNA is then specifically amplified and quantified by qPCR using your miRNA-specific primer and our SYBR Advantage^® qPCR Premix. Multiple miRNA species, as well as the mRNA targets of the miRNAs, can be amplified from a single cDNA sample. The system is extremely sensitive and able to detect miRNAs down to 50 copies.

Highly specific Detection

To demonstrate the specificity of Mir-X miRNA quantification we used a series of 8 highly similar synthetic Let7 miRNA variants (Figure 2). We first spiked each of the Let7 miRNAs into separate samples of yeast poly(A) ^+RNA and generated cDNA using the Mir-X single-tube reaction. We then tested a panel of variant-specific primers with each cDNA sample to determine each primer’s ability to specifically and individually quantify the Let7 subtypes in the cDNA sample. Despite the high degrees of similarity among the variants and the primers (Figure 2A), Mir-X qPCR was highly specific in detecting each Let7 variant (Figure 2B).

For more information, see the Mir-X flyer.

 

 

 

 

Figure 1. Mir-X miRNA qRT-PCR SYBR Kits use a single-step, single-tube reaction to produce first-strand cDNA, which is then specifically and quantitatively amplified using a miRNA-specific primer and SYBR Advantage qPCR chemistry. In the Mir-X cDNA synthesis reaction, RNAs are poly(A)-tailed using poly(A) polymerase, and then copied using a modified oligo(dT) primer and SMART MMLV Reverse Transcriptase.

 

 

Figure 2. Specific quantification of Let7 miRNA variants. Using miRNA-specific primers (Panel A), Mir-X qRT-PCR was able to specifically detect and quantify each member of a series of 8 synthetic Let7 variants that had been spiked into a background of yest poly(A) ⁺ Panel B).The primers detected each of their corresponding Let7 miRNA cognates, but did not detect the off-target variants in 63 of 64 possible combinations.