Digital PCR vs. qPCR: Which One Should You Use? (Pros & Cons)

 

Introduction

The polymerase chain reaction (PCR) has undergone modifications through three generations. The first generation of PCR relies on gel electrophoresis for the analysis of PCR products, yet it faces limitations due to its inadequate detection threshold, labor-intensive procedures, and its singular application scope (qualitative). The subsequent generation of PCR, commonly referred to as real-time quantitative PCR (RT-qPCR), is capable of quantifying products using a standard curve; however, it also exhibits low tolerance to the presence of interfering substances. Digital PCR (dPCR), representing the third generation of PCR, facilitates absolute quantification by partitioning the reaction into discrete compartments.

Characterized by its higher sensitivity and precision in molecular identification, this technology has exhibited applicability in domains such as trace DNA identification, the detection of infrequent mutations, and the assessment of copy number variations.

How the technology Works

Digital Droplet PCR is a refined method of absolute nucleic acid quantification. Unlike qPCR, which gives relative quantification based on fluorescence curves, ddPCR works by partitioning a sample into thousands of nanoliter-sized droplets—each acting as its own PCR micro-reaction.

At the end, you count how many droplets were positive or negative, and from this, you directly calculate the number of target molecules, no standard curve required!

Step-by-Step

Sample Partitioning
Your DNA or cDNA sample is mixed with PCR reagents and divided into ~20,000 droplets using oil emulsion.

PCR Amplification
Each droplet undergoes thermal cycling. Some droplets will contain the target sequence—others won’t.

Fluorescence Detection
After amplification, droplets are passed through a reader that detects fluorescence, telling you which droplets were positive.

Quantification
Using Poisson statistics, ddPCR gives you absolute copy number per microliter of reaction.

🎥 Watch the Full Breakdown

Be sure to check out our YouTube video:
📌 [Digital Droplet PCR Explained | 3rd Generation PCR]

From Total RNA to Pure mRNA | Best Lab Technique

 

Introduction

When working with RNA extracted from complex biological samples—such as tissues, blood, or cell cultures—one common challenge is the presence of highly abundant, non-target RNA. These dominant RNA species, like globin mRNA and ribosomal RNA, can easily overshadow the transcripts you're actually interested in. This is especially relevant in studies involving blood-stage parasites like Plasmodium, where parasite-derived mRNAs are often low in abundance compared to the host RNA background. Other relevant scenarios include studies where a less abundant organism or cell type is present. Without proper RNA enrichment, the signals from these low-abundance transcripts may be lost.

To address this, researchers typically use one of two strategies to enrich for protein coding or target-related  RNA:

-  Enrich for target mRNA using methods that selectively capture polyadenylated transcripts (if applicable), or
- Deplete host globin and ribosomal RNAs using targeted probes or kits.

NEBNext Globin & rRNA Depletion Protocol In Brief:

- 200ul of PCR tubes

- 80% freshly prepared ethanol (10ml 1ml Ethanol + 9ml of nuclease-free water).

- RNA XP cleanup beads

- 0.5-1ug of total RNA as input

- Magnetic Rack

- Esky of ice

1) HYBRIDISATION (on ice)

5ul of total RNA (0.5-1ug)

3ul of NEBNext Globin & rRNA depletion solution

2ul Hybridization mix

5ul nuclease-free water

Thermal cycler setting: 95oC for 2min (with heated lid set to 105oC; Ramp down to 22oC at 0.1oC/sec for 5min; Hold at 4oC

Briefly spin

2) RNase H DIGESTION (on ice)

15ul Hybridized RNA from step above

2ul NEBNext Thermostable RNase H

2ul RNase H Reaction Buffer

1ul Nuclease-free water

total vol. is 20ul

Pipette up-and-down x10 times

Briefly spin down contents

Incubate in pre-heated thermal cycler

Thermal cycler setting: 50oC for 30min (with heated lid set to 55oC; Hold at 4oC

Briefly spin down contents

3) DNase I DIGESTION (on ice) 

(tip: make a mastermix of the reagents if you have more than 1 sample)

20ul RNase H treated RNA

5ul DNase I Reaction Buffer

2.5ul DNase I

22.5ul Nuclease-free water

Total vol. is 50ul

e.g. for x4 preps, mastermix 

22ul of DNase I reaction Buffer

11ul DNase I

99ul Nuclease-free water

Aliquot 30ul of mastermix into individual tubes

Add 20ul of appropriate RNase H treated RNA

Mix up-and-down x10 times

Briefly spin

Incubate in pre-heated. Thermal cycler setting: 37oC for 30min (with lid set to 40oC or off).

Briefly spin.

4) Purification Using RNA XP Clean Up Beads

- Vortex beads

- 90ul of beads to RNA samples

- Pipette up-and-down x10 times

- Incubate tubes for 15min on ice

- Place on magnetic rack until beads collect on side 

- Carefully remove and DISCARD supernatant

- 200ul of 80% ethanol (while on magnetic rack)

- Incubate for 30sec then remove supernatant & discard

- Repeat 80% ethanol wash

- Remove residual ethanol and air dry for 5min

- Add 7ul nculease-free water to lute RNA from beads 

- Pipette up-and-down x10 times

- Briefly spin

- Incubate for 2min

- Place on magnet

- Aspirate 5ul of eluted RNA

Store at -20oC until needed

Watch our video on how mRNA enrichment is typically performed in the lab.

Both approaches help amplify the signal from the parasite and reduce the noise from the host, allowing for a clearer view of the parasite’s gene expression landscape—especially those subtle transcripts that might hold clues to its biology, drug resistance, or lifecycle.

Conclusion

Regardless of the system you're studying, enriching for target RNA or depleting abundant background RNA is a key step in generating meaningful transcriptomic data. By minimising unwanted host RNA, you can amplify the expression signals of interest—whether from pathogens, rare cell types, or low-expression genes—allowing deeper insights into the biology at play.