uMRT Two-Step RT-qPCR Kit

The UltraMarathonRT® Two-Step RT-qPCR Kit is the reverse transcription kit that eliminates a pervasive RT-qPCR artifact across RNA-targeting CRISPR systems. Most kits use conventional retroviral reverse transcriptase (e.g., MMLV RT) to copy RNA into cDNA, but these enzymes lack strand displacement activity, so cDNA synthesis can be directly blocked by the tightly bound guide RNA (gRNA) itself.  With bound gRNA preventing the MMLV RTs from reading through the guide-binding region, RT-qPCR amplicons spanning or sitting just upstream of the gRNA site can be suppressed as a result, artificially indicating knockdown of the target gene. This pervasive effect is observed across RNA-targeting CRISPR systems, including Csm, PspCas13b, and CasRx. 

Key Features and Advantages:

• Accurate CRISPR KO/KD quantitation: UltraMarathonRT's intrinsic helicase reads straight through bound guide RNA, giving you accurate knockdown quantification regardless of system employed or amplicon location.
• Data Reliability: Provides consistent C_t values regardless of primer type (e.g., gene-specific, oligo(dT), or randomer) or location. Other RT-qPCR kits will provide widely different C_t values if primers are in high vs low GC content areas, whereas the UltraMarathonRT® Two-Step RT-qPCR Kit will provide consistent C_t values regardless of location.
• Robust Performance: Achieve exceptional sensitivity and efficiency, ideal for detecting low-abundance RNA, viral RNA, non-coding RNAs, long RNAs, and even highly structured RNAs. This makes it a versatile tool for a wide range of molecular biology applications.
• High Inhibitor Tolerance: Consistent C_t values are maintained in the presence of inhibitory compounds potentially found in biological sample preparation such as ethanol (resistant to 10%), isopropanol (resistant to 5%), TRIzol (resistant to 0.7%), Formalin (resistant to 0.0025%), Ammonium Acetate (resistant to 50mM), Bile Salts (resist to 0.05%), and Hematin (resistant to 10 µM).
• Broad Dynamic Range: Capable of detecting RNA across a wide spectrum of input concentrations ranging from 0.1 pg to 2 µg, providing unparalleled flexibility in experimental design.
• Minimal Experimental Time: An optimized protocol allows researchers to generate complete RT-qPCR data in under 1 hour.
• Preserves RNA Template Integrity: UltraMarathonRT has intrinsic helicase activity for RNA structure unwinding, making the high temperature reaction conditions utilized by other RTs totally unnecessary.
• Dye-Based Detection: Incorporates reliable dye-based technology to facilitate accurate and reproducible real-time monitoring of RNA levels.

Key Applications:

• Evaluation of RNA-targeting CRISPR knockdown (Csm, PspCas13b, CasRx) — eliminates false-positive signal from guide-RNA-blocked RT
• Verification of RNAi and ASO knockdown/knockout
• Also supports RNA-Seq verification, alternative splice variant quantification, RNA degradation measurement, RNA editing quantification, and mutation detection/allele discrimination.
  

Kit Components:

• UltraMarathonRT
• 2x RT Buffer Q
• Nuclease-free Water
• dNTP Mix (10 mM each)
• Random Primers (15mer) (10 µM)
• Oligo(dT)₁₈ Primer (5 μM)
• 2x qPCR Green Dye Master Mix
  

Catalog# R1006S (10 RT rxns: 200 qPCR rxns), R1006M (25 RT rxns: 500 qPCR rxns), R1006L (50 RT rxns: 1,000 qPCR rxns) 

For the product brochure, click here, or for purchase orders, please contact us at sales@rnaconnect.com. 

Reagents required but not supplied: 

• RNA template: 0.1 pg - 2 μg total cellular RNA or 0.1 pg - 500 ng of mRNA are recommended for each reaction.
• PCR Primers: Because uMRT provides full 5’-3’ RNA transcript coverage, PCR amplicon location along uMRT’s full-length cDNA molecule synthesized using gene-specific primer or oligo(dT) primer does not affect qPCR quantification. This greatly reduces the potential for bias and/or C_t value variability observed when using randomers with non-processive retroviral RTs.
• Gene-Specific RT primers.

Storage conditions: For short duration of storage (< 3 month), UltraMarathonRT can be stored in a -20℃ freezer. -80℃ is recommended for long-term storage. All other kit components are stable in a -20℃ freezer for long-term storage. Note that UltraMarathonRT and the 2x qPCR Green Dye Master Mix can endure 10 freeze-thaw cycles without losing noticeable activity. After 20 freeze-thaw cycles, UltraMarathonRT maintains 90% of its original activity.