CRISPR Types Explained: Class, Type and Subtype Made Simple

 

I will be honest with you. The first time I tried to make sense of CRISPR classification, I closed the paper with a headache. Roman numerals, capital letters, “Cas” followed by a number, and what seemed like a new name for every protein. Does it really have to be this complicated? The short answer is no. It looks far worse than it is, and once you see the logic behind it, the whole system falls into place.

In this post I will walk through how CRISPR-Cas systems are classified, what the classification is actually based on, and where the famous Cas9 protein fits in. Along the way I will explain every term as we meet it, so you can follow even if you have never opened a microbiology textbook.

A quick bit of history

The story starts in 1987, long before anyone dreamed of gene editing. A Japanese team led by Yoshizumi Ishino was sequencing a gene in Escherichia coli and noticed something odd next to it: short, repeated DNA sequences separated by unique stretches of DNA. Nobody knew what they were for. For years they remained a curiosity, a puzzle sitting quietly in bacterial genomes.

Then the puzzle began to grow. Similar repeats turned up in other bacteria, and in archaea too. In 2002, Ruud Jansen and colleagues gave them the name we use today, CRISPR (clustered regularly interspaced short palindromic repeats), and noticed that a set of genes always travelled alongside them. They called those genes cas, for CRISPR-associated.

The real breakthrough came in 2007. Rodolphe Barrangou and colleagues, working with the yogurt bacterium Streptococcus thermophilus, showed that these repeats act as an adaptive immune system. When a virus that infects bacteria (a bacteriophage, or phage) attacks, the bacterium can save a small piece of the phage DNA between its repeats. That saved piece is called a spacer. Next time the same phage arrives, the bacterium recognizes it and destroys it. A memory of past infections, written into the genome. Beautiful, isn’t it?

After that, the field exploded, and so did the number of different CRISPR systems being discovered. That is exactly why a classification was needed.

How common is CRISPR in the first place?

Before we sort the systems into boxes, a fair question: how many microbes have one? Genome surveys suggest that roughly 40% of sequenced bacterial genomes carry a CRISPR-Cas system, while about 85% of archaea do. Treat these as rough figures. Databases over-represent bacteria that are easy to grow or medically important, while the many bacteria we cannot culture in the lab are under-represented. CRISPR systems are also gained and lost frequently, so even two strains of the same species can differ. And the exact number shifts a little depending on how a survey defines a system, for example whether a lone CRISPR array without cas genes counts.

What is the classification based on?

Every CRISPR-Cas system does three basic jobs.

• Adaptation: capturing new spacers from invaders. This is done by Cas1 and Cas2.
• Processing: turning the CRISPR array into short guide RNAs, called crRNAs.
• Interference: using a guide RNA to find the invader and destroy or disable it.

Cas1 and Cas2 are found in almost every system, so they cannot tell one system from another. Instead, the classification focuses on the interference step, and in particular on the effector module. The effector is the machinery that recognizes the target and deals with it. It is the part that makes each system different.

The classification then works as a hierarchy, like a family tree with three levels.

1. Class is decided by the architecture of the effector: is it a team of several proteins or a single large protein?
2. Type is decided by a signature gene, one diagnostic cas gene that is found only in that type.
3. Subtype is decided by the finer details, such as extra accessory genes, how the genes are arranged in the locus (the stretch of DNA where they sit), and the evolutionary relationships between the proteins, which is called phylogeny.

Wait, is the effector just the cutter?

Mostly, but not quite. The effector does the whole interference job, and that includes finding the target as well as cutting it. In some systems one protein does both. In others, the two jobs are split between different parts.

Think of it this way. In some systems the effector is a Swiss army knife that finds and cuts. In others it is a crew, where some members locate the target and others do the cutting. The cutter is part of the effector, but it is not the whole story.

Class 1: the team players

Class 1 systems use a multi-subunit effector complex, meaning several Cas proteins assemble together into one machine. They make up roughly 90% of CRISPR loci found in nature.

Type	Signature gene	Target	What to remember
I	Cas3	dsDNA	The most common type overall. A complex called Cascade finds the target, then Cas3 shreds it.
III	Cas10	RNA, and DNA while it is being transcribed	Works only when the target is being transcribed. Uses small signalling molecules called cyclic oligoadenylates.
IV	Csf1	Not well understood	Rare. Mostly found on plasmids and prophages, and often missing adaptation genes.

Type I is a lovely example of divided labour. Cascade is the finder: a guide RNA plus several Cas proteins that scan DNA for a match. Once Cascade locks on, it recruits Cas3, a protein that combines a helicase (an enzyme that unwinds DNA) with a nuclease (an enzyme that cuts nucleic acids). Cascade finds, Cas3 shreds. Type I also needs a short motif next to the target called a PAM (protospacer adjacent motif), which helps the system tell invader DNA from the bacterium’s own.

Type III works differently. Its complex recognizes RNA, and the Cas10 subunit makes cyclic oligoadenylate signals that switch on other nucleases such as Csm6. Because it needs the target to be transcribed, it checks that the invader is actively doing something before it strikes.

Class 2: the lone wolves

Class 2 systems use a single, large, multidomain protein that does everything the Class 1 team does. They are only about 10% of loci, but they are the stars of genome editing, precisely because one protein is so much easier to move into another cell than a whole complex.

Type	Signature gene	Target	What to remember
II	Cas9	dsDNA	Needs a tracrRNA as well as the crRNA. Blunt cut. Two nuclease domains, HNH and RuvC.
V	Cas12	dsDNA and ssDNA	RuvC nuclease only. Staggered cut. Cas12a needs no tracrRNA. Includes the miniature Cas12f.
VI	Cas13	RNA	Two HEPN domains. Collateral RNA cleavage, which is the basis of SHERLOCK diagnostics.

A few of those words deserve a plain explanation. A tracrRNA (trans-activating CRISPR RNA) is a second RNA that Cas9 needs, alongside the crRNA, to become active. HNH and RuvC are the two nuclease domains of Cas9: each one cuts one strand of the DNA double helix. HEPN domains are the RNA-cutting parts of Cas13. And collateral cleavage means that after Cas13 finds its target, it keeps chopping up any nearby RNA, which can be harnessed as a detection signal in a diagnostic test.

Where does Cas9 live?

Cas9 is the signature protein of Type II, and Type II is a minority, roughly 5 to 10% of CRISPR-bearing genomes in most surveys. It is essentially a bacterial protein and is very rare or absent in archaea. It turns up mainly in Firmicutes and Proteobacteria, with scattered occurrences in Bacteroidetes, Actinobacteria, Spirochaetes and Cyanobacteria. Even within one genus it is patchy, so having Cas9 in one species tells you little about its relatives.

The subtypes are defined by accessory genes.

• II-A carries Csn2 alongside Cas9. Examples include Streptococcus pyogenes (the source of SpCas9, the workhorse of the lab), S. thermophilus, Staphylococcus aureus (SaCas9), Listeriaand some Lactobacillus and Enterococcus species.
• II-B carries Cas4. Examples include Legionella pneumophila and Francisella novicida.
• II-C has only Cas1 and Cas2 alongside Cas9. It is the most widespread and diverse subtype, found in Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Treponema denticola, Corynebacterium diphtheriae and others.

Why does anyone care about the different versions? Because Cas9 proteins from different species, called orthologs, differ in size and in the PAM they recognize. SpCas9 is about 1,368 amino acids long and recognizes NGG. SaCas9 is smaller, at about 1,053, and recognizes NNGRRT. CjCas9 is smaller still, at about 984. Smaller orthologs are attractive when you need to fit the editor inside a delivery vehicle with limited space, such as an AAV (adeno-associated virus).

A simple way to remember it all

If the Roman numerals still feel slippery, try this.

• Class tells you one protein or many.
• Type is decided by a CRISPR system’s signature protein. A CRISPR system with Cas3 is Type I, one with Cas9 is Type II, one with Cas10 is Type III, one with Csf1 is Type IV, one with Cas12 is Type V, and one with Cas13 is Type VI.
• Subtype is the letter after the numeral, such as II-A or II-C, and it tells you which variant, mainly from the accessory genes.

And a handy shortcut for spotting Type II: if the system needs a tracrRNA, you are probably looking at Cas9 and not Cas12.

The classification keeps growing

This is not a finished map. Class 2 effectors appear to have evolved from nucleases carried by mobile genetic elements. Cas12, for example, is thought to descend from TnpB-like proteins found in transposons (jumping genes). Each time researchers discover a new family of these proteins, the classification has to stretch to fit it. The 2020 update recognized 2 classes, 6 types and 33 subtypes, and those numbers have likely moved since. More types have since been proposed, which tells you how much of the microbial world is still unexplored.

Why this matters for you

If you work in a lab, or are heading towards one, this classification is more than trivia. It explains why Cas9 needs a tracrRNA and Cas12a does not. It explains why Cas13 is used for RNA detection while Cas9 is used for DNA editing. And it points to where the next generation of tools might come from: the small, strange and little-studied systems sitting in the bacteria we have not yet learned to grow.

Watch the video

Prefer to see it explained? The Adwoa Biotech YouTube channel has a video on the basics of CRISPR-Cas9, covering the history, guide RNAs, PAM sequences and the sickle cell example. https://youtu.be/VlbGQiB83-Y?si=0gnpCu2gmcYZN0cR

Key takeaways

• CRISPR-Cas systems are classified mainly by their effector module, the machinery that recognizes and deals with the target.
• Class 1 uses a multi-subunit complex (Types I, III, IV). Class 2 uses a single protein (Types II, V, VI).
• The type is set by a signature gene, and the subtype by accessory genes, locus organization and phylogeny.
• Cas9 is the signature protein of Type II, found in a minority of CRISPR-bearing bacteria.
• The effector is the whole interference machinery, not just the cutter.

References

Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D. A., & Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science, 315(5819), 1709-1712. https://doi.org/10.1126/science.1138140

Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology, 169(12), 5429-5433. https://doi.org/10.1128/jb.169.12.5429-5433.1987

Jansen, R., van Embden, J. D. A., Gaastra, W., & Schouls, L. M. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Molecular Microbiology, 43(6), 1565-1575. https://doi.org/10.1046/j.1365-2958.2002.02839.x

Makarova, K. S., Haft, D. H., Barrangou, R., Brouns, S. J. J., Charpentier, E., Horvath, P., Moineau, S., Mojica, F. J. M., Wolf, Y. I., Yakunin, A. F., van der Oost, J., & Koonin, E. V. (2011). Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology, 9(6), 467-477. https://doi.org/10.1038/nrmicro2577

Makarova, K. S., Wolf, Y. I., Iranzo, J., Shmakov, S. A., Alkhnbashi, O. S., Brouns, S. J. J., Charpentier, E., Cheng, D., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J. M., Scott, D., Shah, S. A., Siksnys, V., Terns, M. P., Venclovas, C., White, M. F., Yakunin, A. F., ... Koonin, E. V. (2020). Evolutionary classification of CRISPR-Cas systems: A burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67-83. https://doi.org/10.1038/s41579-019-0299-x

Shmakov, S., Smargon, A., Scott, D., Cox, D., Pyzocha, N., Yan, W., Abudayyeh, O. O., Gootenberg, J. S., Makarova, K. S., Wolf, Y. I., Severinov, K., Zhang, F., & Koonin, E. V. (2017). Diversity and evolution of class 2 CRISPR-Cas systems. Nature Reviews Microbiology, 15(3), 169-182. https://doi.org/10.1038/nrmicro.2016.184

Related Content

• CRISPR-Cas9 basics: history, guide RNA, PAM and the sickle cell example https://adwoabiotech.blogspot.com/2026/11/crispr-cas9-explained-guide-rna-pam.html
• Specificity versus sensitivity in CRISPR diagnostics https://adwoabiotech.blogspot.com/2026/11/crispr-diagnostics-specificity.html
• Gene editing delivery and virus-like particles https://adwoabiotech.blogspot.com/2026/11/gene-editing-delivery-why-virus-like.html

RNA Extraction Using Norgen Biotek: A Step-by-Step Laboratory Protocol

If you are extracting RNA from blood in Tempus tubes and you want every size of RNA, including the small species, the Preserved Blood RNA Purification Kit I from Norgen Biotek is worth a close look. It is a spin-column kit made specifically for Tempus tubes, and it is the quickest of the three Tempus-compatible kits to run.

The workflow will feel familiar. You dilute the stabilised blood, pellet the RNA, dissolve the pellet in a guanidinium-based lysis solution, add ethanol & bind to a column. 

Norgen's resin binds RNA depending on the ionic conditions, so proteins pass through or stay on top of the resin, with no phenol or chloroform. The kit also offers an optional on-column DNase step, which is handy when genomic DNA matters.

So how does it run on the bench? Here is the full procedure, step by step.

You'll need

Kit

Norgen Biotek Preserved Blood RNA Purification Kit I, for use with Tempus Blood RNA Tubes (product no. 43400, 50 preps). All components are stored at room temperature and stay stable for at least a year unopened.

• Tempus Blood RNA Tube Diluent
• Lysis Solution
• Wash Solution (supplied as a concentrate)
• Elution Solution
• Mini spin columns
• Collection tubes
• Elution tubes (1.7 mL)

You'll also need

• Blood collected in Tempus Blood RNA Tubes
• 95 to 100% ethanol (and 95% ethanol for the wash solution)
• Sterile 50 mL conical tubes
• Swing-bucket centrifuge that can run at 4 °C
• Benchtop microcentrifuge capable of 14,000 × g, ideally with variable speed
• Vortexer
• Optional: Norgen RNase-Free DNase I Kit (product no. 25710) for the on-column DNase step
• Gloves, lab coat and eye protection, because this is human blood

Part 1: Prepare the Wash Solution

Do this before starting the samples. Add 50 mL of 95% ethanol to the bottle of concentrated Wash Solution. This gives a final volume of 72 mL. Tick the box on the label to show that the ethanol has been added.

Make sure all solutions are at room temperature before use, & work quickly through the whole procedure, because speed protects the RNA.

Part 2: Prepare the lysate

Step 1: Transfer to a conical tube

Pour the entire contents of the Tempus tube into a new 50 mL conical tube.

Step 2: Dilute with Diluent

Add 3 mL of Tempus Blood RNA Tube Diluent, or enough to bring the volume to 12 mL. Close the tube tightly & vortex vigorously for 30 seconds.

Step 3: Pellet

Centrifuge at 4 °C at 3,000 to 5,000 × g (at least 4,500 rpm in a swing-bucket rotor of the Beckman JB-6 type) for 30 minutes. The RNA pellet is transparent and invisible.

Step 4: Drain

Discard the supernatant, then leave the tube inverted on a paper towel for 2 minutes to remove excess liquid.

Step 5: Lyse & add ethanol

Add 600 µL of Lysis Solution to the pellet & vortex for a few seconds to resuspend it. Then add 300 µL of 95 to 100% ethanol & vortex briefly to mix.

Part 3: Bind the RNA to the column

From here on, all centrifugation is done at room temperature in a microcentrifuge at 14,000 × g, unless stated otherwise. The manufacturer warns that temperatures below 15 °C can form precipitates that clog the column.

Step 6: Load

Assemble a column with a collection tube. Apply up to 600 µL of the lysate and ethanol mixture and centrifuge for 1 minute at least 3,500 × g (about 6,000 rpm).

Check that the whole volume has passed through. If not, spin for 1 additional minute at 14,000 × g.

Step 7: Repeat if needed

Discard the flow-through, reassemble the column and collection tube, and repeat Step 6 until all of the lysate has been loaded. With 900 µL of lysate in total, you need two loads.

Part 4: Optional on-column DNase treatment

The kit already gives low genomic DNA carry-over, but the manufacturer provides this step for sensitive downstream work. It is done right after binding, so decide before you start (required if you are doing RNA-Seq). It is particularly worth considering for parasite qRT-PCR.

Step 8: Prepare the DNase

For each column, prepare a mix of 15 µL of RNase-free DNase I and 100 µL of Enzyme Incubation Buffer from Norgen's DNase kit, then mix by inverting. Do not vortex. Apply 100 µL of the solution to each column in Step 10.

Step 9: Pre-wash

Apply 400 µL of Wash Solution to the column and centrifuge for 2 minutes. Discard the flow-through and reassemble the column.

Step 10: Apply and incubate

Apply 100 µL of the DNase solution to the column and centrifuge for 1 minute at 14,000 × g. Pipette the flow-through back onto the top of the column, which is needed for full DNase activity and the best recovery of small RNAs. Incubate the column assembly at 25 to 30 °C for 15 minutes.

Then go straight to the second wash in Part 5 (Wash 2) without any further spin.

Part 5: Wash

Step 11: Wash three times

Add 400 µL of Wash Solution to the column and centrifuge for 1 minute. Check that the full volume passed through, discard the flow-through and reassemble the column. Repeat for a second and third wash. Three washes are needed to clear the salt from the binding step.

Step 12: Dry the resin

Spin the column for 2 minutes to dry the resin thoroughly. This removes traces of ethanol that can interfere with downstream applications. Discard the collection tube.

Wash	Solution	Volume (µL)	Spin
1	Wash Solution	400	1 minute at 14,000 × g
2	Wash Solution	400	1 minute at 14,000 × g
3	Wash Solution	400	1 minute at 14,000 × g
Dry	None	None	2 minutes at 14,000 × g

Part 6: Elute

Step 13: Elute

Place the column in a fresh 1.7 mL elution tube. Add 50 µL of Elution Solution. Centrifuge for 2 minutes at 200 × g (about 2,000 rpm), followed by 1 minute at 14,000 × g. Note the volume eluted. If the full 50 µL has not come through, spin at 14,000 × g for 1 additional minute.

Step 14: Second elution (recommended)

For maximum recovery, repeat Step 13 with another 50 µL of Elution Solution into a separate tube. Keep the two eluates apart, since the second is more dilute.

Store the RNA at −20 °C for a few days, or at −70 °C for the long term.

How do you know it worked?

The manufacturer quotes an average of 5 to 25 µg of RNA from 3 mL of preserved human blood, with RNA of all sizes, including species below 200 nucleotides. One column binds a maximum of 50 µg. With parasitised patient blood, quantify with a fluorometric method such as Qubit or RiboGreen and check the traces on a bioanalyser, since the RNA is a mix of host and parasite RNA.

Troubleshooting tips

• Poor recovery: check that the full volume of Lysis Solution was used, that ethanol was added to both the lysate and the Wash Solution, and that you used the supplied Elution Solution.
• Clogged column: usually caused by incomplete solubilisation of the blood, or by a centrifuge that is cooler than 15 °C during the column spins.
• Degraded RNA: RNase contamination, a procedure that took too long, or poor storage of the purified RNA.
• Poor performance downstream: carry-over of salt (do all three washes) or ethanol (do not skip the dry spin).
• Genomic DNA contamination: use the on-column DNase step, or treat the eluted RNA with RNase-free DNase I.

Reference

Norgen Biotek Corp. (2015). Preserved Blood RNA Purification Kit I (for use with Tempus Blood RNA Tubes) product insert (Product No. 43400, PI43400-3).

RNA Extraction from Tempus Blood Tubes: Tempus Spin RNA Isolation Protocol

When your RNA is locked inside a Tempus tube, you need a protocol that was built for it. The Tempus Spin RNA Isolation Kit is exactly that: the column-based partner to the Tempus Blood RNA Tube, made by the same manufacturer and designed around the same stabilising chemistry.

The principle is simple. The stabilising reagent in the tube lyses the blood cells and precipitates the RNA, while genomic DNA and proteins stay in solution. After a dilution, a long spin pellets that RNA, a short wash and resuspension follow, and the RNA is then cleaned up on a silica-based filter in a microcentrifuge. The manufacturer quotes 6 to 25 µg of RNA from 3 mL of blood, with very low genomic DNA carry-over.

How does it differ from the magnetic bead workflow? There are no beads, no magnet and no DNase step in the manual, so it is a shorter and simpler procedure, but it is manual only and aimed at larger RNA species such as mRNA and rRNA. Here is the full workflow, step by step.

You'll need

Kit

Applied Biosystems Tempus Spin RNA Isolation Kit (cat. 4380204, 50 preps), used with Tempus Blood RNA Tubes (cat. 4342792). All components are stored at room temperature.

• RNA purification filters
• 2 mL collection tubes
• 1× PBS
• RNA Purification Resuspension Solution
• RNA Purification Wash Solution 1
• RNA Purification Wash Solution 2
• Nucleic Acid Purification Elution Solution

You'll also need

• Blood collected in Tempus Blood RNA Tubes
• Sterile 50 mL conical tubes
• Serological pipettes and pipette tips
• Vortexer
• Microcentrifuge capable of 16,000 × g
• Temperature-controlled centrifuge capable of more than 3,000 × g, with a swing-bucket rotor for 50 mL tubes
• Heating block for microcentrifuge tubes, set to 70 °C
• Absorbent paper
• Gloves, lab coat and eye protection, because this is human blood

Part 1: Before you start

Nothing needs to be prepared. The wash solutions and elution solution come ready to use, and the user guide does not call for any additional ethanol.

Two rules apply to the whole procedure. First, the stabilising reagent must be diluted to a final concentration of 1× with Ca²⁺/Mg²⁺-free PBS before extraction, otherwise yields drop significantly. Second, keep the samples on ice as much as possible.

Part 2: Thaw and dilute the blood

Step 1: Thaw (if frozen)

Thaw frozen Tempus tubes at room temperature (18 to 25 °C). Once thawed, keep them on ice.

Step 2: Transfer to a conical tube

Remove the cap and pour the entire contents of the Tempus tube into a clean 50 mL conical tube.

Step 3: Dilute with 1× PBS

Add 3 mL of 1× PBS (Ca²⁺/Mg²⁺-free) to bring the total volume to 12 mL. If the tube held less than 3 mL of blood, add enough PBS to make up the difference to 12 mL.

Cap the tube properly, then vortex at maximum vortex speed for 30 seconds. Vortexing for less time can clog the filter later. A layer of froth on top is normal.

Part 3: Pellet the crude RNA

Step 4: Pellet

Centrifuge at 4 °C at 3,000 × g for 30 minutes. The RNA pellet is transparent and invisible, so handle the tube gently.

Step 5: Decant and drain

Carefully pour off the supernatant without dislodging the pellet. Leave the tube inverted on absorbent paper for 1 to 2 minutes, then blot the drops of liquid from the rim with clean absorbent paper.

Part 4: Resuspend the RNA

Step 6: Add Resuspension Solution

Add 400 µL of RNA Purification Resuspension Solution, placing the pipette tip at the bottom of the tube so you do not wash blood residue down the inside wall. Vortex briefly to resuspend the pellet.

The resuspended RNA can wait on ice while you set up the next part.

Part 5: Bind and wash on the filter

Between each step, discard the liquid in the waste tube and put the filter back. The manufacturer notes that you can also move the filter into a fresh collection tube each time.

Step 7: Set up and pre-wet

Label an RNA purification filter and insert it into a waste collection tube. Pre-wet the membrane with 100 µL of RNA Purification Wash Solution 1.

Step 8: Load the RNA

Add about 400 µL of the resuspended RNA to the filter and centrifuge for 30 seconds at 16,000 × g.

Step 9: Wash

Wash once with Wash Solution 1 and twice with Wash Solution 2, as laid out in the table below. Then give the membrane a final spin to dry it.

Wash	Solution	Volume (µL)	Spin
1	RNA Purification Wash Solution 1	500	30 seconds at 16,000 × g
2	RNA Purification Wash Solution 2	500	30 seconds at 16,000 × g
3	RNA Purification Wash Solution 2	500	30 seconds at 16,000 × g
Dry	None	None	30 seconds at 16,000 × g

Part 6: Elute

Step 10: First elution

Move the filter to a new, labelled collection tube. Add 100 µL of Nucleic Acid Purification Elution Solution, close the cap, and incubate the entire tube for 2 minutes at 70 °C. Then centrifuge for 30 seconds at 16,000 × g.

Step 11: Re-elute

Add about 100 µL of the collected eluate back onto the filter and centrifuge for 2 minutesat 16,000 to 18,000 × g. No incubation is needed this time.

Step 12: Collect the RNA

Discard the filter. Transfer about 90 µL of the eluate to a new, labelled tube, taking the liquid from the top so you do not disturb the last 10 µL or the pellet at the bottom. Do not throw away the eluate!

Store the RNA at −20 °C, or at −80 °C for the long term.

How do you know it worked?

The manufacturer reports an A260/A280 ratio above 1.9, genomic DNA below 0.05% by weight, and 6 to 25 µg of RNA from 3 mL of blood. With parasitised patient blood, the RNA is a mix of host and parasite RNA, so measure it with a fluorometric method such as Qubit or RiboGreen and check the traces on a bioanalyser rather than relying on the A260/A280 ratio alone.

Unlike the MagMAX kit, this procedure has no DNase step. For gDNA-sensitive work such as qRT-PCR of parasite genes, include no-RT controls, and consider a DNase treatment after elution. That treatment is not part of the manufacturer's protocol.

Troubleshooting tips

• Black or brownish-red filters: the blood was not mixed well with the stabilising reagent after collection. Shake or vortex the tube for 10 to 20 seconds immediately after the draw. If residue got onto the filter, add an extra 500 µL of Wash Solution 1 and another 500 µL of Wash Solution 2, with a 30 second spin at 16,000 × g after each.
• Degraded RNA: residual protein with RNase activity, or lysate exposed to more than 37 °C. Add extra wash steps next time until the membrane looks white, and vortex for the full 30 seconds after the PBS dilution.
• Leaks during vortexing: the conical tube was not capped properly.
• Clogged filter: usually caused by too little vortexing after dilution, or by microclots from poor mixing at the time of blood collection.

Reference

Thermo Fisher Scientific. (2018). Tempus Blood RNA Tube and Tempus Spin RNA Isolation Kit user guide (Publication No. 4379232, Rev. E). Life Technologies Corporation.

How to Culture Plasmodium falciparum Field Isolates: Step-by-Step Protocol

From Patient to Petri Dish: Culturing Plasmodium falciparum from Field Isolates

In 1976, William Trager and James Jensen managed something malaria researchers had chased for decades. They kept Plasmodium falciparum alive outside the human body, growing continuously in nothing more exotic than a flask of red blood cells and culture medium. It sounds almost too simple. It wasn't.

Fifty years later, that same basic system, blood cells, medium, a warm incubator, still underpins how we study malaria in the lab. But there's a meaningful difference between growing a laboratory-adapted strain like 3D7 and coaxing a parasite fresh out of a patient's bloodstream into stable, continuous culture. Lab strains have already adapted to artificial conditions. Field isolates haven't. They arrive stressed, genetically diverse, and often reluctant to grow.

Here's what that process actually looks like, from the moment blood leaves a patient to the point where a flask of parasites is thriving on its own.

Why Field Isolates Are Different

A laboratory clone has been in culture for years, sometimes decades. It's been selected, whether researchers intended it or not, for the traits that make it easy to grow: tolerance of artificial media, resilience to handling, a forgiving growth rate.

A field isolate has none of that history. It comes straight from a patient, carrying whatever genetic background it evolved under in the wild, and it has to survive both the trauma of collection and the abrupt shift from a human host to a plastic flask. Some isolates adapt within days. Others take weeks. Some never do.

This is why timing matters so much at the start. Blood should be collected before antimalarial treatment begins, because drugs in the patient's system will still be acting on the parasites in vitro, and the sample needs to be processed and into culture as quickly as possible.

Collecting and Preparing the Sample

The process starts with a straightforward venous draw, typically a few milliliters into a tube containing an anticoagulant like ACD-A or CPD-A. From there, the sample is split.

One portion goes toward culture. The other, a smaller aliquot, is frozen immediately as a stabilate, essentially an insurance policy. If the culture fails to establish, or if something goes wrong downstream, the frozen stabilate preserves that original isolate for a second attempt later.

The culture-bound portion gets diluted substantially, roughly twenty-fold, in complete culture medium. Diluting this heavily isn't arbitrary. It brings the cell concentration down to something the culture system can actually support, and it happens as soon as possible after collection, because parasite viability starts declining the moment blood leaves the body.

Setting Up the Culture

Once diluted, the isolate goes into flasks under conditions that mimic, as closely as a plastic vessel can, the environment inside a human blood vessel: 37°C, a gas mixture low in oxygen and enriched in carbon dioxide, and a growth medium supplemented with serum-replacement additives like Albumax.

From here the culture is maintained by daily medium changes. Spent medium is aspirated gently, so as not to disturb the settled red cells, and replaced with fresh, pre-warmed medium. The flask is gassed briefly, sealed, and returned to the incubator. Do this every day, at roughly the same time if possible, and a viable isolate will slowly establish itself.

Parasitaemia, the proportion of red cells actually infected, is checked regularly by preparing a thin blood smear, staining it with Giemsa, and counting under oil immersion. When it climbs into the 6 to 8 percent range, the culture is diluted back down to around 1 percent with fresh uninfected red cells. Go much lower than that and, oddly, the culture can stall rather than thrive. There's a floor below which dilution does more harm than good.

Contamination is the other constant threat. A flask that's cloudy, foul-smelling, or simply behaving oddly should be discarded without hesitation. Aseptic technique, correctly, and every time, is the only thing standing between a healthy culture and a ruined one.

Getting the Stages to Line Up

Left alone, a culture will contain parasites at every stage of the 48-hour asexual cycle simultaneously, rings, trophozoites, and schizonts all mixed together. For many experiments that's a problem, because you need parasites at a known, uniform stage.

Sorbitol synchronization solves this by exploiting a quirk of parasite biology: infected red cells become more permeable as the parasite matures, so exposing a culture to a sorbitol solution selectively lyses the later-stage cells and leaves the young rings untouched. Run it once and you've narrowed the population. Run it again, roughly one cycle later, and the synchrony tightens further, since by then the survivors have all matured into rings together too.

Percoll gradient centrifugation takes a different approach, using density rather than membrane permeability to physically separate mature, hemozoin-laden schizonts from the younger stages, which is particularly useful when you need to enrich for schizonts specifically rather than simply narrow the age range.

Banking the Isolate

A field isolate that survives long enough to establish is worth preserving, both for future experiments and because recollecting from the same patient usually isn't possible.

Freezing follows a fairly gentle protocol built around glycerol as a cryoprotectant, added slowly and at room temperature to give it time to penetrate the cells before the drop to liquid nitrogen. Thawing reverses the process in careful stages, using a series of buffered solutions added dropwise to avoid osmotic shock, which would otherwise rupture the very cells you're trying to recover.

Done correctly, a stabilate frozen this way can sit in liquid nitrogen for years and still be revived into a thriving culture.

What Makes It Worth the Trouble

Culturing lab-adapted clones is, by comparison, almost easy. Culturing field isolates is slower, less predictable, and demands more attention at every step. But it's also the only way to study parasites as they actually exist in circulation, carrying whatever drug-resistance mutations, growth characteristics, or genetic diversity they picked up in the population they came from.

That's the whole point. A lab strain can tell you how P. falciparum behaves in principle. A field isolate tells you how it's behaving right now, in the patients walking into a clinic today.

Workflow Overview

Culturing Plasmodium falciparum field isolates can be divided into eight stages.

1. Prepare incomplete RPMI 1640.
2. Prepare complete RPMI 1640.
3. Prepare supporting reagents.
4. Process donor red blood cells.
5. Screen donor blood for contamination.
6. Establish parasite cultures.
7. Perform daily culture maintenance.
8. Monitor parasite growth and dilute cultures when required.

Each of these stages is described in detail below.

Before You Begin 

Successful parasite culture depends as much on preparation as it does on technique. Before beginning, ensure that all media have been prepared and filter sterilised, the incubator has reached 37°C, and all tissue culture flasks and complete RPMI have been pre-warmed. Maintaining sterile technique throughout the procedure is essential because bacterial or fungal contamination can rapidly destroy a parasite culture.

Field isolates are often less tolerant of environmental stress than long-established laboratory strains. Consequently, minimise the amount of time cultures spend outside the incubator and handle culture flasks gently to avoid disturbing the sedimented erythrocytes.

🧪 Adwoa Biotech Insight

One of the simplest ways to improve parasite recovery is to organise your workspace before removing cultures from the incubator. Arrange your pipettes, sterile tips, microscope slides and fresh medium in advance so that routine maintenance can be completed quickly while the parasites remain close to their optimal growth temperature.

Materials and Equipment 

Reagents 

• RPMI 1640 powder (Gibco)

• Albumax

• HEPES

• Dextrose

• Hypoxanthine

• Sodium bicarbonate

• Sodium hydroxide (NaOH)

• Hydrochloric acid (HCl)

• Distilled water

• Sodium chloride

• Sorbitol

• Glycerol

• Methanol

• Giemsa stain

• Giemsa buffer

• Immersion oil

Equipment 

• Class II Biological Safety Cabinet

• Magnetic stirrer

• pH meter

• Refrigerated centrifuge

• 0.2 μm filter unit

• 25 cm² tissue culture flasks

• 75 cm² tissue culture flasks

• Incubator maintained at 37°C

• Light microscope with a 100× oil immersion objective

• Micropipettes and sterile pipette tips

• Sterile Pasteur pipettes

• Glass microscope slides

Preparing Incomplete RPMI 1640

Incomplete RPMI 1640 forms the foundation of the parasite culture medium. It supplies amino acids, vitamins, inorganic salts and nutrients required for parasite growth while allowing additional supplements to be added during preparation of the complete culture medium. Because every culture established in the laboratory depends on this solution, careful preparation is essential.

Reagents 

Reagent	Quantity
RPMI 1640 powder (Gibco)	10.4 g (1 packet)
HEPES	7.15 g
Dextrose	2 g
Hypoxanthine (25 mg/mL in NaOH)	2 mL
Distilled water	To 1 litre

Procedure 

1. Add approximately 850 mL of distilled water to a large sterile conical flask.
2. Place the flask on a magnetic stirrer and slowly dissolve one packet (10.4 g) of RPMI 1640 powder.
3. Add 7.15 g HEPES and continue stirring until dissolved.
4. Add 2 g dextrose.
5. Add 2 mL hypoxanthine (25 mg/mL prepared in NaOH).
6. Continue stirring until the solution becomes completely clear.
7. Adjust the pH to 7.2 using sodium hydroxide or hydrochloric acid.
8. Top up the solution with distilled water to a final volume of 1 litre.
9. Filter sterilise through a 0.2 μm membrane filter inside a biological safety cabinet.
10. Store the medium at 4°C for up to two months.

🧪 Adwoa Biotech Insight

Each component of incomplete RPMI serves a specific purpose. HEPES helps maintain a stable pH while cultures are handled outside the incubator, dextrose provides an additional source of glucose for parasite metabolism, and hypoxanthine supplies purines that Plasmodium falciparum cannot synthesise itself. Preparing the medium accurately and maintaining the correct pH are essential for achieving consistent parasite growth.