Choosing glass fiber filters for nucleic acid extraction

Glass fiber filters are a popular and effective choice for nucleic acid extraction applications. But how do you choose the best grade for your application? In this blog, we explore how glass fiber filter grades can change your nucleic acid yield.

Due to its high nucleic acid binding capacity, silica-containing materials — such as silica coated superparamagnetic beads and glass fiber filters — are widely used as a binding material for nucleic acid extraction in commercial kits. But, with a range of glass fiber (GF) filters available, how do you choose the best grade for your application?

Learn more about using glass fiber filters for nucleic acid extraction

Whether you’re a researcher, molecular diagnostic developer, or kit manufacturer, when designing and optimizing a nucleic acid extraction method, it is important to consider your final application. Often, one of the key differences to consider is the size of the target DNA.

So, does the grade of glass fiber filter really matter when it comes to nucleic acid extraction? And should the size of the target DNA influence your choice of glass fiber filter? We decided to investigate using a range of GF filters (Table 1)!

Table 1. Characteristics of Whatman™ glass fiber filters from Cytiva

Extracting DNA: Does size matter?

We first explored how these different grades of glass filters (Table 1) performed for four sizes of DNA fragment:

• 3Gb DNA from human blood
• 50 Mb DNA from tomato leaves
• 10 Kb DNA plasmid
• 1.7 Kb PCR product

We standardized the tests using a commercially available DNA extraction kit, substituting the column and binding material for different glass filter grades (Fig 1). To serve as a comparison, we used the unaltered commercially available column alongside our glass fiber filter columns, taking samples from the same lysate.

Fig 1. Experimental setup: Nucleic acid-extraction of different DNA/RNA fragment sizes using various grades of Whatman™ filters available from Cytiva.

By UV spectrophotometry, we quantified the DNA yield for each sample. Interestingly, all Whatman™ glass filter membranes outperformed the commercial column for the larger DNA fragment sizes (Fig 2A and 2B). In both cases, the MG1 glass fiber filter yielded the most DNA.

Conversely, when it came to smaller DNA fragments, the commercial column yielded more DNA than the MG1 glass fiber filters. But, both GF/F and GF/B grades performed similar to or better than the control (Fig 2C and 2D).

Fig 2. DNA yields from solid-phase extractions using different grades of glass fiber filters as the solid phase binding material for (A) 3 Gb Human blood, (B) 50 Mb Tomato leaves, (C) 10 Kb Plasmid, and (D) 1.7 Kb PCR product. Mean average of three batches of each filter grade, with five repeats conducted for each batch, plotted for each sample type.

For a follow-up test, we wanted to find out whether GF/F and GF/B grade glass filters are suitable for even smaller fragments. We used the same experimental set-up as before but this time we extracted DNA and RNA, with the former sheared to produce fragments that simulate the sizes present in cell-free DNA/RNA (cfDNA/cfRNA) of human plasma (~50 bp).

Again, GF/F and GF/B filters resulted in greater yields of nucleic acids compared to the commercial column (Fig 3).

Fig 3. DNA yields from solid-phase extractions using different grades of glass fiber filter for (A) simulated cfDNA and (B) RNA. Mean average of three batches of each filter grade, with five repeats conducted for each batch, plotted for each sample type.

So, does size matter? When choosing a glass filter grade for nucleic acid extraction, the answer appears to be yes!

Discover our range of Whatman™ glass fiber filter membranes

Maximum loading capacity: Getting more bang for your buck!

The maximum loading capacity of a filter will influence its recovery rate, which is the amount of nucleic acid recovered versus input.

Since the GF/F filter grade generated the highest yields of DNA for the PCR fragment as well as the highest yields of nucleic acids for the simulated cfDNA/cfRNA, we investigated its maximum loading capacity and recovery rates relative to the commercially available spin column (Fig 4).

Fig 4. Comparison of maximum loading capacities of Whatman™ GF/F and commercial kit filters. (A) Extraction amount at different sample loading amounts and (B) Recovery rate at different sample loading amounts.

We found that the maximum loading capacity and recovery rates were considerably higher for the GF/F filter compared to the commercially available spin column. For example, the commercially available spin column yielded approximately 30 µg of DNA from a sample containing 50 µg; equating to a recovery rate of 60%. Comparatively, the GF/F filter recovered >90% of the initial 50 µg starting material.

As you might expect, increasing the sample loading amount did also increase the extraction, but with diminishing returns. While this might be inefficient, if your sample is in abundance, it could help maximize the yield in the given elution volume.

Conclusions on nucleic acid extraction using glass fiber filters

Our short study demonstrates that Whatman™ glass fiber filters from Cytiva enable nucleic acid yields from extractions that are comparative to or exceed a commercially available spin column.

Our tests indicated that the efficiency of nucleic acid extraction was influenced by size of fragment and choice of filter grade, with Whatman™ GF/F glass fiber filters being particularly well suited for extracting small fragments.

Consequently, for developers, we would suggest that it’s important to optimize the nucleic acid extraction protocol in light of its final application; taking into account the size of target fragments. Nevertheless, the availability of glass filter material in easy-to-use formats provides flexibility for assay design and optimization at a competitive price point. For example, “punch-and-go” discs allow for layered stacking of the same or different grades of glass fiber to achieve optimal nucleic acid binding, loading capacity, and yield.

Cytiva provides a broad portfolio of glass fiber filters in a range of formats that can support nucleic acid extraction applications. Flexibility in formats and grades is key to development and optimization of nucleic acid extraction methods for startups right through to established companies, regardless of stage in research and development or scale.

Alternative to glass fiber filters

Magnetic beads (or superparamagnetic particles) are versatile little tools for easy and effective isolation of biomolecules. There are many types of magnetic beads available. Different surface coatings and chemistries give each type of bead its own binding properties, which can be used for magnetic separation (isolation and purification) of nucleic acids, proteins, or other biomolecules in an easy, effective, and scalable way.

This ease-of-use makes them automation friendly and well suited for a range of applications, including sample preparation for next generation sequencing (NGS) and PCR, protein purification, molecular and immunodiagnostics, and even magnetic activated cell sorting (MACS), among many others.

If you need support with any aspect of nucleic acid extraction, would like to find out more about our product range, or to request a sample, please contact your local Cytiva representative or the Scientific Support team. We’re always here to help!

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