SYBR Green I Assay for Malaria Drug Testing: Step by Step

Here is a fact I never get tired of: the cell that malaria lives in has thrown away its own DNA. Mature human red blood cells have no nucleus and no genome, so there is nothing in them to stain. That means any DNA you find in a Plasmodium falciparum culture belongs to the parasite. And what does that allow us to do? If we can make DNA glow, we can count parasites, and if we can count parasites, we can ask how much of a drug it takes to stop them.

For a long time, answering that question meant working with radioactivity. Labs fed parasites a radiolabelled substrate and measured how much they took up, which demanded special equipment, safety procedures and a lot of patience. Then, in 2004, Smilkstein and colleagues reported a simple, inexpensive, one-step alternative built on SYBR Green I, a dye whose fluorescence rises sharply when it binds double-stranded DNA (Smilkstein et al., 2004). No isotopes, no wash steps. Just a plate reader. 

In this post I walk through the whole assay, from the drug dilution plate to the IC50, using the layout from my video. It is a 96-well format, so it scales nicely from a single compound to a full screen.

What the assay actually measures

SYBR Green I barely fluoresces on its own. Slip it between the strands of DNA and it lights up, roughly a thousand (1000) times brighter. After 72 hours of growth, you lyse the cells, add the dye, and read the plate. More parasites mean more DNA, which means more green. Add an effective drug and the signal falls. Plot fluorescence against the logarithm of the drug concentration, fit a curve, and the midpoint is your IC50.

One small note on timing. The original paper measured growth after 48 hours, while many labs now use 72 hours so that the parasites go through more than one cycle and slower-acting drugs have a fair chance to show an effect. Pick one, and keep it identical across experiments.

Step 1: Build the drug plate

Prepare your drugs at twice the final concentration in complete medium, because they will be diluted one to one when the parasites go in. Dissolve compounds in DMSO if needed, and keep the final DMSO at or below 0.2% in every well, including the controls.

Add 50 µL of complete medium to the wells that will carry the lower concentrations. Put 100 µL of the top 2× concentration in the first drug column, then transfer 50 µL to the next column, mix, and carry on across the plate, discarding the last 50 µL. Every drug well now holds 50 µL, and each column is half the concentration of the one before. That is your two-fold serial dilution.

Then plan the controls. Edge wells evaporate faster and give odd readings, so fill them with 100 µL of water or PBS and do not use them for parasites. In my layout, columns 2 to 10 of rows B to D hold the test compound, rows E to G hold chloroquine as a reference drug, and column 11 holds the controls: no-drug parasites for 100% growth, and uninfected red cells for background.

Step 2: Prepare the parasite suspension

Hematocrit is the percentage of the culture volume that is packed red cells. We want 2% in the well. Because the parasites are mixed one to one with the drug, you prepare the suspension at 4% hematocrit and the well ends up at 2% in a total volume of 100 µL.

Do you need to add uninfected blood? Only if your culture is above the parasitaemia you want. Parasitaemia is the fraction of red cells that are infected, so the infected cells are already part of the hematocrit and are not counted twice. If your culture is already at 0.5% to 1%, spin it at 500 × g for 5 minutes, remove the supernatant, and use the pellet as your packed cells. If it is higher, dilute the pellet with uninfected red cells until you reach the target parasitaemia.

Here is a bulk mix for one plate, with a little extra for pipetting losses:

Component	Volume (5.5 mL batch at 4% hematocrit)
Packed infected red cells (pellet)	220 µL
Complete medium	5.28 mL

Make a small separate batch of uninfected red cells at 4% hematocrit for the background wells, from the same donor and batch you used to dilute the parasites. That way the background matches the cells in the rest of the plate.

Step 3: Combine and incubate

Add 50 µL of the parasite suspension to each drug well and to the no-drug wells, and 50 µL of the uninfected suspension to the background wells. Each well now holds 100 µL at 2% hematocrit. Place the plate in a humid chamber, flush it with a low-oxygen gas mixture (5% O₂, 5% CO₂, 90% N₂), seal it, and incubate at 37 °C for 72 hours. This is a good time to go and do something else. The plate does not need you.

Step 4: Lyse and stain

The lysis buffer does two jobs. Saponin and Triton X-100 open the red cells and the parasite membranes, and EDTA keeps nucleases quiet, so that the dye can reach the DNA. Prepare it in advance, and add the dye only on the day.

Component	Final concentration
Tris, pH 7.5	20 mM
EDTA	5 mM
Saponin	0.008% (w/v)
Triton X-100	0.08% (v/v)
SYBR Green I (10,000× stock)	0.2 µL per mL of lysis buffer (about 2×)

This is the recipe from the original paper, and it works well for most labs (Smilkstein et al., 2004). SYBR Green I is light sensitive, so protect the buffer with foil. Add 100 µL to every well and mix until no visible red cell sediment remains. Then wrap the plate in foil and leave it for 1 hour at room temperature in the dark.

Step 5: Read the plate

Use a fluorescence plate reader with excitation at about 485 nm and emission at about 530 nm. Black plates reduce crosstalk between wells. After three days of waiting, the reading takes about a minute, which feels almost rude.

Step 6: From fluorescence to IC50

First, subtract the mean of the uninfected red cell wells from every reading. Second, set the mean of the no-drug wells to 100% growth. Third, plot the percentage growth against the log of the drug concentration and fit a sigmoidal dose-response curve with nonlinear regression, in GraphPad Prism or a similar program. The concentration at the midpoint of the curve is your IC50.

Run each concentration in triplicate, and repeat the whole experiment on at least two or three separate days. Technical replicates tell you how well you pipette, while independent experiments tell you whether the result is real. And check your reference drug: if the chloroquine IC50 for a well-characterised strain is far from the values reported in the literature, the assay is telling you something, and it is not about your compound.

Things that bite you

• White blood cells. They carry DNA too. This is no problem for cultured parasites, but it matters for fresh patient blood unless you remove the leukocytes first.
• DNA-binding compounds. A drug that grabs DNA competes with the dye and can fake a result, and a coloured compound can quench the signal.
• Slow-acting drugs. Some compounds need a longer exposure than 72 hours to show their full effect.
• Edge wells. Fill them with water or PBS, not parasites.
• Light and age. Protect the dye from light and make the final buffer fresh.

Watch the video

Prefer to see it done? I walk through the whole assay, step by step, in the video below, including the plate layout and the dose-response curve.

Video: SYBR Green I Assay: Malaria Drug Testing Step by Step

Related Content

• Hematocrit calculation for passaging P. falciparum
• The P. falciparum life cycle
• Giemsa stain: morphology and history
• Red blood cell isolation protocol

References

Smilkstein, M., Sriwilaijaroen, N., Kelly, J. X., Wilairat, P., & Riscoe, M. (2004). Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrobial Agents and Chemotherapy, 48(5), 1803-1806. https://doi.org/10.1128/AAC.48.5.1803-1806.2004

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.

MagMAX RNA PURIFICATION FROM TEMPUS TUBES

Column-based RNA kits are reliable, but they ask a lot of your centrifuge & your patience. Magnetic bead kits take a different route. The RNA sticks to tiny paramagnetic beads, you hold the beads against a magnet, & you wash & elute without a single spin column in sight.

The Invitrogen MagMAX for Stabilized Blood Tubes RNA Isolation Kit (compatible with Tempus Blood RNA Tubes, cat. 4451893) applies this idea to 3 mL of whole blood that has been stabilised in a Tempus tube. The manufacturer quotes roughly 3 to 25 µg of total RNA, including microRNA, per tube. The kit supplies a 96-well processing plate, so you can work in plates or in plain 1.5 mL tubes, depending on how many samples you have.

So what does the workflow actually look like at the bench? Below is the manual, tube-based version, step by step.

You'll need

Kit

Invitrogen MagMAX for Stabilized Blood Tubes RNA Isolation Kit, compatible with Tempus Blood RNA Tubes (cat. 4451893, 96 preps)

• Room temperature: Tempus Resuspension Solution, Binding Solution Concentrate, Wash Solution 1 Concentrate, Wash Solution 2 Concentrate, Elution Buffer, 96-well Processing Plate
• 2 to 8 °C (do not freeze): Tempus Pre-Digestion Wash, RNA Binding Beads, Tempus 1× PBS
• −15 to −25 °C: Tempus Proteinase, TURBO DNase

You'll also need

• Blood collected in Tempus Blood RNA Tubes (3 mL per tube)
• 100% isopropanol and 100% ethanol
• RNase-free 50 mL conical tubes
• Non-stick RNase-free 1.5 mL microcentrifuge tubes (or a 96-well plate and orbital shaker)
• Magnetic stand that fits your tubes or plate
• Swing-bucket centrifuge with 50 mL tube adapters, capable of 5,000 × g at 4 °C
• Vortexer with a microtube adapter, & a microcentrifuge
• Aerosol-resistant, nuclease-free pipette tips, RNase decontamination solution & absorbent paper
• Gloves, lab coat eye protection, because this is human blood

Part 1: Prepare the wash solutions

Do this before starting the samples. Each RNA isolation uses 300 µL of each prepared wash solution in total (two 150 µL washes each).

Wash Solution 1

Add 8 mL of 100% isopropanol to each bottle of Wash Solution 1 Concentrate. Mix by inverting 3 to 4 times, then tick the label so nobody forgets that the isopropanol is already in.

Wash Solution 2

Add 24 mL of 100% ethanol to the bottle of Wash Solution 2 Concentrate. Mix by inverting 3 to 4 times and mark the label in the same way.

Both can be stored at room temperature until needed.

Part 2: Thaw & dilute the blood

Keep the samples on ice as much as possible. The manufacturer warns that yields can drop significantly if the samples warm up.

Step 1: Thaw (if frozen)

Thaw frozen Tempus tubes on ice for 10 to 30 minutes.

Step 2: Transfer to a conical tube

Label a clean 50 mL conical tube and pour in the entire contents of the Tempus tube.

Step 3: Dilute with Tempus 1× PBS

Add 3 mL of Tempus 1× PBS to bring the total volume to 12 mL. If your tube held less than 3 mL of blood, add enough PBS to make up the difference, because the stabilising reagent must be at a final concentration of 1×. Cap the tube securely and vortex vigorously for 30 seconds. Frothing is normal.

Move straight on to the next part. Do not leave the diluted blood sitting around.

Part 3: Pellet and wash the crude RNA

Step 4: Pellet

Centrifuge the conical tubes at 5,000 × g for 15 minutes at 4 °C. The crude RNA pellet is transparent, so you will not see it. Handle the tubes gently so you do not dislodge it.

Step 5: Decant & wash

Pour off the supernatant carefully. The manufacturer recommends decanting rather than pipetting, because a pipette can suck up the invisible pellet.

Mix the Tempus Pre-Digestion Wash by gently inverting the bottle 3-4 times, then add 4 mL to each conical tube. Re-cap and vortex at moderate speed for 5 seconds.

Step 6: Re-pellet & drain

Centrifuge again at 5,000 × g for 10 minutes at 4 °C. Pour off the supernatant, then invert the tubes on absorbent paper for 2 minutes. Blot the tube rims with clean absorbent paper, and place the tubes on ice.

Part 4: Resuspend and digest

Step 7: Make the resuspension mix

Prepare this just before use. Per sample you need 117.5 µL Tempus Resuspension Solutionplus 2.5 µL Tempus Proteinase, which gives 120 µL. The manufacturer advises including 5% extra to cover pipetting losses. Mix by vortexing at moderate speed, spin down for 2 to 3 seconds, and keep it on ice.

Samples	Resuspension Solution (µL)	Proteinase(µL)	With 5% excess
2	235	5	246.8 µL solution + 5.3 µL proteinase
4	470	10	493.5 µL solution + 10.5 µL proteinase
6	705	15	740.3 µL solution + 15.8 µL proteinase
8	940	20	987 µL solution + 21 µL proteinase
12	1,410	30	1,480.5 µL solution + 31.5 µL proteinase

Step 8: Resuspend the pellet

Add 120 µL of the mix to the bottom of each conical tube, re-cap, and vortex gently for 5-10 seconds. Transfer the whole volume to a labelled 1.5 mL tube (or one well of the 96-well plate).

Stopping point: the resuspended crude RNA pellets can sit on ice for up to 4 hours.

Step 9: DNase digestion

Add 10 µL TURBO DNase to each tube, then mix at vigorous speed for 10 minutes. This is done at room temperature (18 to 25 °C). If your lab is colder than 18 °C, the manufacturer suggests running the digestion in a 25 °C incubator.

Part 5: Bind the RNA to the beads

Work at room temperature from here on. Cap tubes before vortexing, & give them a 2-3 second spin after each mixing step to collect the liquid.

Step 10: Add beads & Binding Solution

Vortex the RNA Binding Beads at vigorous speed until they are fully resuspended. To each sample add 50 µL Binding Solution Concentrate and 20 µL of the vortexed beads. Mix at moderate speed for 1 minute.

Step 11: Add isopropanol

Add 200 µL of 100% isopropanol & mix at moderate speed for 3 minutes. Order matters here: the beads must go in before the isopropanol, or yields suffer.

Step 12: Capture the beads

Place the tubes on the magnetic stand for about 1-3 minutes, until the solution clears & the beads form a pellet at the magnet. Aspirate & discard all the supernatant without disturbing the beads. Then take the tubes off the magnet, which the manufacturer marks as critical before the next step.

Part 6: Wash

Step 13: Wash Solution 1 (twice)

Add 150 µL Wash Solution 1, mix at moderate speed for 1 minute, and capture the beads on the magnet for about 1 to 2 minutes. Aspirate the supernatant, remove the tubes from the magnet, and repeat once.

Step 14: Wash Solution 2 (twice)

Repeat exactly the same routine with 150 µL Wash Solution 2: mix for 1 minute, capture, aspirate, remove from the magnet, and repeat once.

Part 7: Dry and elute

Step 15: Dry the beads

Open the tubes and invert them on absorbent paper for 2 minutes at room temperature. If you can see residual Wash Solution 2, remove it with a fine-tipped pipette (without touching the beads) and invert for 1 more minute. Do not over-dry: over-dried beads are harder to resuspend.

Step 16: Elute

Add 80 µL Elution Buffer and mix at vigorous speed for 4 minutes. You can use anywhere from 20 to 80 µL, depending on how concentrated you want the RNA. Capture the beads for 1 to 3 minutes, then transfer the supernatant, which holds your RNA, to a nuclease-free tube. Do not throw it away!

Use the RNA straight away from ice, or store it at −20 °C for up to 6 months, or at −80 °C for the long term.

How do you know it worked?

Measure the concentration by absorbance at 260 nm. The manufacturer expects an A260/A280 ratio of 1.8 to 2.2 and notes that RNA outside that range can still work in qRT-PCR. For a more reliable picture, use a bioanalyzer: a 28S to 18S ratio of 1.0 or above is considered acceptable for RNA from blood in Tempus tubes, and the RIN gives a fuller view of degradation.

Troubleshooting tips

• Low yield: check that the tube was filled to the black mark, that the PBS dilution reached 12 mL, and that you vortexed for the full 30 seconds.
• Lost pellet: decant the supernatant slowly rather than pipetting, and use a swing-bucket rotor.
• Beads clumping at elution: incubate at 70 °C for 5 minutes and repeat the 4-minute shaking step. Preheating the Elution Buffer to 70 to 80 °C also helps.
• Brownish eluate: beads have been carried over. Put the tube back on the magnet and transfer the RNA to a fresh tube.
• Poor downstream performance: check that the correct alcohol went into each wash concentrate and that the beads were fully dry before elution.

Reference

Applied Biosystems. (2010). MagMAX for Stabilized Blood Tubes RNA Isolation Kit (compatible with Tempus Blood RNA Tubes) protocol (Part No. 4452007, Rev. A). Life Technologies Corporation.