Real-time quantitative PCR (RT-qPCR) is a molecular biology technique for the real-time amplification and detection of specific DNA or RNA sequences. This method uses fluorescent dyes or probes that emit signals when bound to amplified DNA or RNA molecules, enabling quantification of target sequences. At its core, fluorescent quantitative PCR detects and quantifies PCR amplification products via fluorescent probes or DNA-binding dyes and a real-time qPCR instrument, which measures fluorescence during thermal cycling.

Methods to Improve the Sensitivity of RT-PCR Reaction Systems

1. Isolate High-Quality RNA

Successful cDNA synthesis requires high-quality RNA. High-quality RNA must be full-length, free of reverse transcriptase inhibitors such as EDTA or SDS, and contain minimal genomic DNA contamination. RNA quality determines the maximum sequence information that can be transcribed into cDNA.

2. Additives to Promote Reverse Transcription

Additives including glycerol and DMSO can be included in first-strand synthesis reactions to reduce nucleic acid duplex stability and unwind RNA secondary structures. Up to 20% glycerol or 10% DMSO can be added without compromising M-MLV activity. AMV can also tolerate up to 20% glycerol without reduced activity.

3. Increase Reverse Transcription Incubation Temperature

Higher incubation temperatures help unwind RNA secondary structures and increase reaction yield. For most RNA templates, incubating RNA and primers at 65°C in the absence of buffer or salt, followed by rapid cooling on ice, eliminates most secondary structures and allows primer annealing.

4. Use Reverse Transcriptases Lacking RNase H Activity

RNase inhibitors are often added to reverse transcription reactions to increase cDNA length and yield. Both M-MLV and AMV possess endogenous RNase H activity in addition to their polymerase activity. RNase H competes with polymerase for RNA:DNA heteroduplexes and degrades the RNA strand in RNA:DNA complexes. RNA templates degraded by RNase H can no longer serve as effective substrates for cDNA synthesis, reducing yield and length. Eliminating or significantly reducing RNase H activity in reverse transcriptases is therefore highly beneficial.

5. RNase H Treatment

Treating cDNA synthesis reactions with RNase H prior to PCR can improve sensitivity. For some templates, residual RNA in cDNA reactions blocks amplicon binding; RNase H treatment resolves this and increases sensitivity. RNase H treatment is typically required when amplifying long, full-length cDNA targets. For most RT-PCR reactions, RNase H treatment is optional, as the 95°C PCR denaturation step usually hydrolyzes RNA in RNA:DNA complexes.

6. Enhance Detection of Small RNA Quantities

RT-PCR is particularly challenging with limited RNA quantities. Adding RNase-free glycogen as a carrier during RNA isolation helps increase yield from small samples. RNase-free glycogen can be added simultaneously with Trizol. For samples from <50 mg tissue or <10⁶ cultured cells, the recommended concentration of RNase-free glycogen is 250 μg/mL.

7. Reduce Genomic DNA Contamination

A potential challenge in RT-PCR is genomic DNA contamination in RNA preparations. Treating RNA with amplification-grade DNase I prior to reverse transcription removes contaminating DNA. Terminate DNase I digestion by incubating samples at 65°C for 10 minutes in 2.0 mM EDTA. EDTA chelates Mg²⁺, preventing Mg²⁺-dependent DNA hydrolysis at high temperatures.

To distinguish amplified cDNA from contaminating genomic DNA, design primers spanning introns so that cDNA amplicons are shorter than those from genomic DNA. Additionally, perform a no-reverse-transcriptase control for each RNA template to determine whether a given fragment originates from genomic DNA or cDNA. PCR products obtained in the absence of reverse transcriptase derive from genomic DNA.

ROCGENE | Archimed Series Real-Time Quantitative PCR Instruments

A successful experiment relies on high-performance detection instruments. The Archimed series qPCR instruments are the world’s first time-resolved real-time fluorescent quantitative PCR systems, meticulously developed by Rocgene’s international senior technical team by integrating the essence of quantitative PCR technology. Featuring a novel Fresnel lens optical system, patented time-resolved signal acquisition, and unique temperature control, Archimed delivers higher detection sensitivity, superior thermal accuracy and uniformity, streamlined operation, and comprehensive analytical capabilities. With 1-copy detection sensitivity and 1.33× resolution, it enhances low-concentration sample detection. Backed by global design and manufacturing standards, Archimed achieves international-level quality.

About Rocgene

Founded in 2016, Rocgene (Beijing) Scientific Instruments Co., Ltd. is a high-tech biotech company driven by core technologies and established by scientists and business professionals. Rocgene is committed to translating cutting-edge life science research into clinically relevant products, developing proprietary research and molecular diagnostic tools.

Rocgene has been honored as a National High-Tech Enterprise, Beijing Specialized, Sophisticated, Unique, and New Enterprise, and Zhongguancun High-Tech Enterprise. It holds 4 Class III medical device registration certificates, 10 Class I medical device filings, 6 CE certifications, 3 US FDA clearances, 3 UK MHRA certifications, and 3 Indonesia NADFC certifications. The company has 67 patents (41 invention, 12 utility model, 14 design) and 47 software copyrights. Rocgene products are exported to nearly 20 countries worldwide.

In July 2023, Rocgene relocated to its Beijing headquarters, housing a >10,000 m² R&D and operations center with standardized laboratories. Its Xuzhou subsidiary operates a >10,000 m² intelligent manufacturing center with 4 instrument production lines and 3 reagent lines. The company is ISO 13485 and ISO 9001 certified and continuously improves quality management.

With the courage to transcend and ambition to lead, Rocgene strives to become a forward-looking industry pioneer.