MystiCq® microRNA RT-qPCR Workflow Process

MystiCq microRNA reagents provide a complete SYBR Green RT-qPCR workflow for quantifying microRNA expression in four steps: isolation, polyadenylation, cDNA synthesis and quantitation by amplification.
 

 Using Quantitative PCR to Analyze microRNA Expression Levels

MystiCq Workflow

Step 1: microRNA (or total RNA) isolation

As with any RT-qPCR method, it starts with sample isolation.  Because microRNAs are small, standard RNA purification kits focusing on total RNA may not actually preserve small RNAs.  We offer three different products for isolation of microRNAs from a variety of biological sources:

Each product offers its own set of features and benefits based on time, sample type, and the types of RNA being isolated.

Steps 2 and 3: microRNA polyadenylation and cDNA synthesis

Once microRNA is isolated, like any RNA, it needs to be reverse transcribed (RT) into a cDNA template before amplification. Due to their small size and lack of poly-A tail, traditional methods for RT are often incompatible.

MystiCq microRNA cDNA Synthesis Mix (MIRRT) includes everything needed to convert microRNAs into suitable cDNA templates for qPCR. Other MystiCq microRNA cDNA Synthesis Mix benefits include 1) a wide, linear dynamic range of input RNA; 2) incorporation of separate polyadenylation and RT steps so proper background level controls may be used; and 3) conversion of all microRNAs, allowing future qPCR analysis of yet-to-be-discovered microRNAs.

Step 4: microRNA amplification & quantitation

Quantitative PCR is performed using MystiCq microRNA qPCR Primers, Universal PCR Primer (MIRUP-500RXN), MystiCq microRNA SYBR Green qPCR ReadyMixes (MIRRM00/01/02/03)), and cDNA produced using MystiCq microRNA cDNA Synthesis Mix. Each microRNA-specific Assay Primer is specifically designed to target mature microRNA. Search our pre-designed and wet lab tested MystiCq microRNA qPCR Primers to find your microRNA of interest (link coming). Used in conjunction with the Assay Primer for amplification, the Universal PCR Primer binds a region in the cDNA template integrated during the RT process. MystiCq microRNA SYBR Green qPCR ReadyMixes are 2X concentrated, ready-to-use qPCR mixes for use on a variety of real-time PCR platforms. Find the right mix for your equipment here.