Description

This one-step 2× multiplex RT-PCR Master Mix is designed for real-time qualitative and quantitative amplification with TaqMan probes directly from extraction-free RNA samples. The 2× premixed solution provides all necessary components except gene-specific primers, TaqMan probes, and RNA templates. RT-PCR 149 is optimized for high-GC templates and stem-loop rich regions, and supports multiplex detection of up to two targets, ensuring reliable reverse transcription and amplification of challenging RNA templates.

Kit Characteristics

Direct-from-sample: enables expression analysis directly from cultured cells without RNA purification.
Fast preparation: 5-minute lysis including DNase treatment, versus 30–60 minutes for traditional RNA purification.

The master mix contains extra more redundant amounts of the RTase and DNA polymerase, specifically designed to overcome endogenous inhibition in extraction-free RNA samples.
For the reverse transcription step, this kit uses a highly efficient Thermophilic Reverse Transcriptase (US patent pending), which is a thermophilic type A polymerase, with optimal temperatures of 60-62°C.
The RTase is easily heat-inactivated at ≥90°C for 1min.
The RTase efficiently synthesizes a complementary DNA strand on RNA template from a gene-specific primer, ≤1 unit per 20μL of reaction.
The RTase reversely-transcribes single digit copies of target RNA molecules consistently.
The kit also contains Taq-Fast DNA polymerase which extends more than 300 bases with short cycling program.
The concentrations of the primers are variable depending on assay designs and thermocycling protocols (Table 1).
The preferable PCR product size is ≤150bp.
The kit has three formulations of No ROX, Low ROX or High ROX concentrations for your choice.

Kit Components

2X Master Mix (2 × 1 mL / 5 × 1 mL / 10 mL for 200, 500, or 1000 × 20 μL reactions).
Lysis Buffer (2 × 10 mL / 1 × 50 mL / 1 × 100 mL).
User Manual.

Storage

Store at -20°C (stable up to 24 months with ≤10 freeze-thaw cycles). Transport at ≤4°C for up to 3 days.

Reaction Setup

20 μL Reaction: 10 μL Master Mix, primers, ≤4 μL RNA sample, water to 20 μL.
10 μL Reaction: 5 μL Master Mix, primers, ≤2 μL RNA sample, water to 10 μL.
Recommended amplicon size: ≤150 bp.

Compatible Instruments

Compatible with all major real-time PCR instruments, including but not limited to Applied Biosystems®, Bio-Rad®, Roche®, Qiagen®, Eppendorf®, and more.

Workflow

Harvest 10,000–100,000 cells.
Add lysis buffer and incubate for 5 minutes.
Heat at 95°C for 10 minutes.
Add lysate directly to RT-PCR setup.
Run RT-qPCR and analyze results.

Product Comparison

142: SYBR Green, No multiplexing, No probe system, Standard single-gene expression.

146: TaqMan, Yes multiplexing, Yes probe system, Multiplex TaqMan assays.

149: TaqMan, Yes (Super Multiplex), Yes probe system, Highly complex multi-target detection.

Q: What nucleotides are included in the 2× Probe qPCR Master Mix? Does it contain UTP?

A: The 2× Probe qPCR Master Mix contains all standard deoxynucleotides—dATP, dTTP, dGTP, and dCTP—each at a standard concentration of 200 µM.
UTP is not included by default, but custom formulations with UTP can be provided on request, depending on the required quantitative range.
Additionally, the mix uses a next-generation hot-start enzyme, which allows for higher reaction temperatures and results in enhanced dNTP stability and overall amplification performance.

Documents

User Manual

Extraction-free RT-PCR from cultured cells

1-Step 2X Super multiplex RT-PCR Master Mix-TaqMan Probe (#149)-Biofargo