How to Remove Globin and rRNA from Blood RNA Samples: A Complete Guide

 cDNA Synthesis with Globin RNA Depletion: Remove Blood Contamination Before cDNA

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You've extracted RNA from your blood samples. You've checked the concentration and purity (A260/A280 ratio looks good at ~1.8- 2.0). Everything looks perfect. You're ready to synthesise cDNA and start your gene expression analysis.

But then you run your samples and the results are... disappointing. Your lowly expressed genes barely show up. Your data looks noisy. 

What went wrong?

The culprit is probably hiding in plain sight: globin messenger RNA. It's so abundant in blood samples or samples handled in the presence of blood, that it drowns out everything else you're trying to study. It's like trying to hear a whisper in a room full of people shouting.

Today, we're going to solve this problem once and for all. We'll explore why globin RNA is such a nuisance, how to get rid of it, and walk through the exact protocol for globin and ribosomal RNA depletion that will clean up your samples and give you the clean, reliable data you deserve.

🎥 Want to See It in Action?
Check out our video tutorial on cDNA Synthesis with GLOBIN RNA DEPLETION: Remove Blood Contamination Before cDNA on the Adwoa Biotech YouTube Channel, where we walk through the entire protocol step by step.

Total RNA to Globin and rRNA depleted RNA

The Problem: When Abundance Becomes a Nuisance

Let's start with the basics. When you extract total RNA from blood samples or from cells cultured with any amount of blood, you're getting a complex mixture of different RNA types. And not all of them are created equal in terms of abundance.

What's Actually in Your RNA Sample?

Think of your RNA sample like a big bucket of different colored marbles. You want to study the rare, interesting marbles (the lowly expressed messenger RNAs that tell you about specific genes). But your bucket is absolutely stuffed with two types of marbles that you don't care about:

Marble type 1: Ribosomal RNA
These make up about 80% to 90% of your total RNA. They're the structural components of ribosomes, the protein-making machines in your cells. They're everywhere, and they're not telling you anything about gene expression (1).

Marble type 2: Globin messenger RNA
If you're working with blood samples, these marbles completely dominate the remaining space. Globin mRNA can make up 70% or more of the messenger RNA in blood (2-5).

So here's the math: You've got 80% to 90% ribosomal RNA, and then of the remaining 10% to 20%, about 70% of that is globin mRNA. That means the genes you actually want to study might represent less than 5% of your total RNA.

Good luck finding a signal in all that noise.

Why Is Globin mRNA So Abundant?

Here's a quick biology refresher. Red blood cells are packed with hemoglobin, the protein that carries oxygen throughout your body. Hemoglobin is made of four globin protein chains paired with heme groups (the iron-containing part that actually binds oxygen).

The instructions to make these globin chains come from genes located on chromosomes 11 and 16. These genes are transcribed into messenger RNA, which then gets translated into globin proteins. The chains are named with Greek letters: alpha, beta, delta, gamma, epsilon, zeta, and theta.

Because red blood cells need so much hemoglobin to do their job, they produce massive amounts of globin mRNA. And when you extract RNA from blood, all of that globin mRNA comes along for the ride, whether you want it or not (2-5).

The Real-World Impact

Imagine you're trying to study a plasmodium parasite gene expression in a blood sample. Maybe you're looking at virulence gene production or drug resistance markers. Those messenger RNAs are present at very low levels compared to globin (12).

When you synthesise cDNA and run RNA sequencing or qPCR, the globin mRNA sequences dominate. They eat up your sequencing reads. They compete for primers and enzymes. They create background noise that makes it nearly impossible to detect subtle changes in your genes of interest.

It's like trying to have a conversation at a rock concert. The music (globin mRNA) is so loud that you can't hear what anyone is saying (your target genes).

The solution? Turn down the music. Remove the globin mRNA before you even start your cDNA synthesis.

The Solution: Selective RNA Depletion

The elegant solution to this problem is called RNA depletion. Instead of trying to enrich for the RNAs you want (which is tricky and can introduce bias), you specifically remove the RNAs you don't want (6).

The Strategy: Hybridisation and Degradation

Here's how it works:

Step 1: Target the unwanted RNAs with DNA probes

You use short, single-stranded DNA probes that are designed to be complementary to the sequences of globin mRNA and ribosomal RNA. Think of these probes like molecular Velcro. They stick specifically to globin and ribosomal RNA sequences because their bases pair up perfectly (A with T, G with C) (13).

When you mix your total RNA with these DNA probes and heat them up, the probes find their matching RNA targets and bind tightly. This creates RNA-DNA hybrids.

Step 2: Destroy the hybrids with RNase H

Now here's the clever part. There's an enzyme called RNase H that has a very specific job: it recognises RNA-DNA hybrids and chews up the RNA strand, leaving the DNA probe intact (8).

So your globin mRNA and ribosomal RNA, now paired with DNA probes, get completely degraded by RNase H. Meanwhile, your precious messenger RNAs from other genes are left alone because they're not bound to any DNA probes.

Step 3: Clean up the leftovers

After RNase H does its job, you need to remove the DNA probes and any remaining DNA contamination. You use DNase I, an enzyme that degrades DNA. This ensures your final sample contains only the RNA you want, with no DNA contamination that could interfere with downstream applications.

Step 4: Purify the cleaned RNA

Finally, you use magnetic beads to purify your RNA. The beads bind RNA, you wash away all the degraded bits and enzymes, and you elute your clean, depleted RNA ready for cDNA synthesis.

What Gets Removed?

Let's be specific about what this protocol removes:

Globin messenger RNAs removed:
HBA1, HBA2, HBB, HBD, HBM, HBG1, HBG2, HBE, HBQ1, and HBZ. These are all the major globin mRNA variants that clutter up blood samples (13).

Cytoplasmic ribosomal RNAs removed:
5S, 5.8S, 18S, 28S, plus the internal transcribed spacers (ITS) and external transcribed spacers (ETS). These are the ribosomal RNA species that make up the bulk of total RNA in any cell.

Mitochondrial ribosomal RNAs removed:
12S and 16S. These are the ribosomal RNAs from your mitochondria, which also contribute to background noise.

After depletion, what you're left with is enriched messenger RNA from all the other genes in your sample. Your lowly expressed genes now have a chance to be detected (13).

Why Not Just Use Poly(A) Selection?

You might be wondering: Can't I just select for messenger RNAs using their poly(A) tails instead?

Poly(A) selection is another common method where you use oligo(dT) beads to capture messenger RNAs by their poly(A) tails. This naturally depletes ribosomal RNA (which doesn't have poly(A) tails) and most globin mRNA (6).

Here's the catch: some globin mRNAs still have poly(A) tails, so you can end up with globin contamination even after poly(A) selection. If you're working with blood samples, the depletion method we're describing today is more thorough.

Additionally, if you want to study non-polyadenylated RNAs or degraded RNAs, poly(A) selection won't work. Depletion methods give you more flexibility (6).

Understanding the Key Components

Before we get into the protocol, let's quickly review what's in the kit and what each component does.

What's in the NEB Next Globin and Ribosomal RNA Depletion Kit?

NEBNext Globin and rRNA Depletion Solution
This contains the single-stranded DNA probes that target globin mRNA and ribosomal RNA. These are the molecular seekers that find and mark your unwanted RNAs for destruction.

Probe Hybridization Buffer
This creates the right chemical environment for the DNA probes to bind to their RNA targets. Think of it as the matchmaker that brings the probes and targets together.

Thermostable RNase H
This is the enzyme that degrades RNA in RNA-DNA hybrids. It's thermostable, meaning it works at higher temperatures (around 50°C), which helps it work efficiently and reduces non-specific degradation.

RNase H Reaction Buffer
Provides the optimal pH and salt conditions for RNase H to do its job.

DNase I
This enzyme degrades any DNA in your sample, including the DNA probes and any genomic DNA contamination. You don't want DNA hanging around when you're trying to study RNA.

DNase I Reaction Buffer
Creates the right conditions for DNase I activity.

Nuclease-Free Water
Ultra-pure water with no enzymes that might degrade your RNA.

What You Need to Provide

The kit doesn't include everything. You'll need:

• Freshly prepared 80% ethanol (make it fresh every time)

• A microcentrifuge

• A vortex

• A thermal cycler (programmable)

• A magnetic rack for separating beads

• A Bioanalyzer or TapeStation for quality control

• 200 µL PCR tubes

• RNA Sample Purification Beads
  These magnetic beads bind RNA and allow you to wash away all the enzymes, buffers, and degraded material. Store in the fridge, not frozen.

Sample Requirements: Getting Your RNA Ready

Not all RNA samples are created equal. Before you start this protocol, your RNA needs to meet certain quality standards.

RNA Quality Assessment

Run your RNA on an Agilent Bioanalyzer using an RNA 6000 Nano or Pico chip. This gives you the RNA Integrity Number (RIN), which tells you how intact your RNA is. Degraded RNA gives poor results no matter how well you deplete it.

RNA Must Be Free of Contaminants

Your RNA needs to be clean. Specifically:

No salts: Magnesium salts and guanidinium salts (common in extraction buffers) interfere with enzymatic reactions. These need to be removed during purification.

No organic solvents: Phenol and ethanol from extraction need to be completely gone. Residual phenol is particularly nasty and will inhibit enzymes.

No DNA contamination: Genomic DNA is a common hitchhiker in RNA preps. It can come from the interphase during TRIzol extraction or from overloading silica columns during purification.

Why DNA Contamination Is a Special Problem

Here's the tricky part. You need to remove genomic DNA before starting the depletion protocol because genomic DNA can mess up your results. The standard way to do this is to treat your RNA with DNase I before depletion.

But here's the catch: you're also going to use DNase I as part of the depletion protocol to remove the DNA probes. If there's any residual DNase I activity left over from your pre-treatment, it will degrade the single-stranded DNA probes you're about to add, and the whole protocol fails.

So if you treat your RNA with DNase I beforehand, you absolutely must remove the enzyme afterward. You can do this by:

• Phenol-chloroform extraction followed by ethanol precipitation, or

• Column-based purification (like using a silica spin column)

After purification, your RNA must be resuspended in nuclease-free water, not in any buffer.

RNA Input Amount

You can use anywhere from 10 nanograms to 1 microgram of total RNA. It needs to be in a volume of 5 µL of nuclease-free water.

Quantify it accurately using an RNA-specific fluorometric method like Qubit, RiboGreen, or a Bioanalyzer. Don't rely on UV absorbance (NanoDrop) alone because it can overestimate due to contaminants.

The Complete Protocol: Step by Step

Alright, now let's walk through the entire protocol from start to finish. Keep your samples on ice throughout the process.

Step 1: Probe Hybridisation (Binding DNA Probes to Target RNAs)

What you're doing:
Mixing your total RNA with DNA probes that specifically recognise globin mRNA and ribosomal RNA. Then heating and cooling the mixture in a controlled way to allow perfect base pairing.

What you need:

• 5 µL of total RNA (10 ng to 1 µg in nuclease-free water)

• Globin and rRNA Depletion Solution (3 uL)

• Probe Hybridization Buffer (2 uL)

• 5 uL nuclease-free water

The process:

Make a master mix if you're processing multiple samples. Combine the Globin and rRNA Depletion Solution with the Probe Hybridization Buffer and nuclease-free water. Mix this master mix thoroughly by pipetting up and down at least 10 times. It's important to mix well because the probes need to be evenly distributed.

Add 10 µL of your master mix to 5 µL of your total RNA sample for a total volume of 15 µL.

Mix again by pipetting up and down at least 10 times.

Briefly spin the tube in a microcentrifuge just to collect all the liquid at the bottom. You don't want droplets on the side of the tube.

Thermal cycling program:

Now place your tube in a thermal cycler and run this program:

1. Heat to 95°C for 2 minutes (this denatures everything, breaking apart any secondary structures so probes can access their targets)

2. Ramp down to 22°C at a rate of 0.1°C per second (this slow cooling allows the DNA probes to find and bind perfectly to their RNA targets)

3. Hold at 22°C for 5 minutes (this stabilizes the RNA-DNA hybrids)

4. Hold at 4°C until you're ready for the next step

This program takes about 15 to 20 minutes total.

After the program finishes, briefly spin down the tube to collect any condensation, then immediately place it on ice.

Why this step matters:

The heating step ensures that all RNA secondary structures are melted, giving the DNA probes full access to their target sequences. The slow, controlled cooling allows specific, stable hybridization. If you cool too fast, you get non-specific binding. If you cool too slow, you waste time. The 0.1°C per second rate is optimized for maximum specificity.

Step 2: RNase H Digestion (Destroying the Targeted RNAs)

What you're doing:
Adding RNase H enzyme to specifically degrade the RNA portions of the RNA-DNA hybrids you just created. This chews up globin mRNA and ribosomal RNA while leaving your other messenger RNAs intact.

What you need:

• 15 µL of hybridized RNA (from Step 1)

• Thermostable RNase H enzyme

• RNase H Reaction Buffer

• Nuclease-free water

The process:

To your 15 µL of hybridized RNA, add:

• 2 uL of Thermostable RNase H enzyme

• 2 uL of RNase H Reaction Buffer

• 1 uL of Nuclease-free water to bring the total volume to 20 µL

Mix thoroughly by pipetting up and down at least 10 times.

Briefly spin down to collect everything in the bottom of the tube.

Place the tube in a pre-heated thermal cycler and incubate:

• 30 minutes at 50°C

• Lid temperature set to 55°C (to prevent condensation)

• Then hold at 4°C

After incubation, briefly spin down again to collect any condensation, then place on ice. Proceed immediately to the next step.

Why this step matters:

RNase H is incredibly specific. It only degrades RNA when it's base-paired with DNA. Your free-floating messenger RNAs (the ones you want to keep) are completely safe. Only the globin and ribosomal RNAs that are bound to DNA probes get destroyed.

The 50°C temperature is optimal for this thermostable version of the enzyme. It works efficiently and helps reduce any non-specific activity.

Step 3: DNase I Digestion (Removing DNA Probes and Contamination)

What you're doing:
Now that RNase H has destroyed the targeted RNAs, you need to get rid of all the DNA in your sample. This includes the DNA probes themselves and any genomic DNA contamination that might still be present.

What you need:

• 20 µL of RNase H-treated RNA (from Step 2)

• DNase I enzyme

• DNase I Reaction Buffer

• Nuclease-free water

The process:

To your 20 µL of RNase H-treated sample, add:

• 5 µL of DNase I Reaction Buffer

• 2.5 µL of DNase I enzyme

• 22.5 µL of nuclease-free water

This brings your total volume to 50 µL.

Mix thoroughly by pipetting up and down at least 10 times.

Briefly spin down.

Incubate in a thermal cycler:

• 30 minutes at 37°C

• Lid can be off, or if on, set to 40°C

After incubation, briefly spin down and proceed immediately to purification.

Why this step matters:

You absolutely do not want any DNA in your final RNA sample. DNA contamination will interfere with downstream applications like cDNA synthesis and RNA-seq. By treating with DNase I, you ensure that only RNA remains.

The DNase I also destroys the DNA probes, which have now served their purpose. They've marked the unwanted RNAs for destruction, and now they need to go.

Step 4: RNA Purification with Magnetic Beads (Cleaning Up Your Depleted RNA)

What you're doing:
Using magnetic beads to bind and purify your RNA, washing away all the enzymes, buffers, and degraded material. What you elute at the end is clean, depleted RNA ready for downstream use.

What you need:

• 50 µL of DNase I-treated sample (from Step 3)

• RNA Sample Purification Beads (from the fridge, not frozen)

• Freshly prepared 80% ethanol

• Nuclease-free water

• Magnetic rack

The process:

First, vortex the RNA Sample Purification Beads to resuspend them completely. These beads settle at the bottom of the tube, so you need to make sure they're evenly mixed.

Add 90 µL of beads to your 50 µL sample. This is a 1.8x ratio (90 µL beads to 50 µL sample), which is optimal for RNA binding.

Mix thoroughly by pipetting up and down at least 10 times.

Incubate the tubes on ice for 15 minutes. This gives the RNA time to bind to the beads. Don't skip this step or rush it.

After incubation, place the tubes on a magnetic rack. The magnetic field pulls the beads (now bound to RNA) to the side of the tube. Wait until the solution becomes clear. This usually takes about 5 minutes.

Carefully remove and discard the supernatant without disturbing the beads. The beads contain your RNA, so be gentle.

Ethanol washing (very important):

While the tube is still on the magnetic rack, add 200 µL of freshly prepared 80% ethanol. Let it sit for 30 seconds. This washes away salts and contaminants.

Carefully remove and discard the supernatant.

Repeat this wash one more time (for a total of two ethanol washes).

After the second wash, completely remove all residual ethanol. Use a small pipette to get every last drop if necessary.

Let the beads air dry for about 5 minutes. You want the ethanol completely evaporated, but don't over-dry the beads or they'll be hard to resuspend.

Eluting the RNA:

Remove the tube from the magnetic rack.

Add 7 µL of nuclease-free water directly to the beads.

Mix thoroughly by pipetting up and down at least 10 times. This resuspends the beads and allows the RNA to come off into the water.

Briefly spin down the tube.

Let the tube sit at room temperature for 2 minutes. This gives the RNA time to fully elute from the beads.

Place the tube back on the magnetic rack and wait until the solution is clear.

Carefully transfer 5 µL of the supernatant (this contains your purified, depleted RNA) to a new nuclease-free tube.

Your RNA is now ready!

Why this step matters:

The magnetic bead purification is critical for removing all the junk (enzymes, degraded RNA fragments, salts, buffers) from your sample. The ethanol washes are particularly important. They remove salts that would otherwise inhibit downstream enzymes.

The 80% ethanol concentration is optimal. Pure ethanol doesn't wash as effectively, and lower concentrations can cause RNA to come off the beads prematurely.

What You Can Do With Your Depleted RNA

Your RNA is now depleted of globin mRNA and ribosomal RNA. It's clean, pure, and ready for action. Here's what you can use it for:

Random Primed cDNA Synthesis

This depleted RNA is perfect for random primed cDNA synthesis (not oligo(dT) based synthesis). Random primers will reverse transcribe all your remaining messenger RNAs, giving you a complete cDNA library representing all the genes you care about, without the overwhelming background of globin (13).

RNA Sequencing (RNA-Seq)

RNA-seq is one of the most powerful applications. By depleting globin and ribosomal RNA beforehand, you ensure that your sequencing reads go to the genes that matter. Instead of wasting 70% to 90% of your reads on globin and ribosomal sequences, you get deep coverage of lowly expressed genes, better detection of rare transcripts, and more statistical power to detect differential expression.

qPCR and Gene Expression Analysis

If you're doing targeted gene expression studies with qPCR, starting with depleted RNA means you're working with cleaner templates. You get better amplification efficiency, lower background, and more reliable quantification.

Other Downstream RNA Applications

Basically, any application that requires high-quality messenger RNA will benefit from depletion. Northern blots, microarrays, in situ hybridization—all of these work better when you're not fighting through a cloud of globin and ribosomal RNA.

Storage and Handling

Short-term storage:
If you're using your depleted RNA within a few days, store it at -20°C.

Long-term storage:
For longer storage, keep it at -80°C. RNA is stable for months to years at -80°C if stored properly.

Avoid freeze-thaw cycles:
Every time you freeze and thaw RNA, you risk degradation. Aliquot your RNA into small volumes so you only thaw what you need each time.

Common Ribosomal RNA Species: A Quick Reference

Since we're depleting ribosomal RNA, it's worth understanding what we're actually removing. Here's a quick breakdown:

Cytoplasmic Ribosomal RNAs

5S rRNA:
Part of the large ribosomal subunit. It's one of the smaller ribosomal RNAs and plays a structural role in ribosome assembly.

5.8S rRNA:
Approximately 160 nucleotides long, also part of the large ribosomal subunit. It helps stabilize the ribosome structure.

18S rRNA:
Part of the small ribosomal subunit. It's about 1,870 nucleotides long and plays a key role in binding mRNA during translation.

28S rRNA:
Part of the large ribosomal subunit with approximately 5,000 nucleotides. It's involved in catalyzing peptide bond formation.

ITS (Internal Transcribed Spacers):
These are sequences found in the precursor 45S pre-rRNA transcript. They're about 200 to 3,000 nucleotides long and get removed during ribosome assembly. But they can still be present in your RNA prep as processing intermediates.

ETS (External Transcribed Spacers):
These are found at the 5' and 3' ends of the 45S pre-rRNA transcript, ranging from 200 to 2,000 nucleotides. Like ITS, they're removed before the ribosome is fully assembled, but their transcripts can contaminate your RNA sample.

Mitochondrial Ribosomal RNAs

12S rRNA:
Part of the small mitochondrial ribosomal subunit. Mitochondria have their own ribosomes, separate from the cytoplasmic ones.

16S rRNA:
Part of the large mitochondrial ribosomal subunit.

All of these ribosomal RNAs are abundant, non-informative for gene expression studies, and get removed by this depletion protocol.

Tips for Success

Here are some pro tips to make sure this protocol works perfectly every time:

Always use fresh 80% ethanol.
Ethanol absorbs water from the air over time, so the concentration changes. Make it fresh each time you run the protocol.

Mix thoroughly at every step.
This protocol emphasizes mixing (pipetting up and down at least 10 times) for a reason. Incomplete mixing means incomplete reactions, which means incomplete depletion.

Keep samples on ice.
RNA is sensitive to degradation. Keeping everything cold between steps helps preserve it.

Don't over-dry the beads.
When you're air-drying the beads after ethanol washes, stop as soon as the ethanol has evaporated. Over-dried beads are hard to resuspend and you'll lose RNA.

Pre-heat your thermal cycler.
Don't put your samples into a cold thermal cycler and wait for it to heat up. Pre-heat it to the target temperature first for more accurate and consistent incubations.

Use nuclease-free everything.
Tubes, water, tips - everything needs to be RNase-free. One contaminated reagent can ruin your entire experiment.

Troubleshooting Common Issues

Problem: Poor depletion efficiency (globin or rRNA still present)

Possible causes:

• Insufficient mixing during hybridization

• Thermal cycler temperature inaccuracies

• Degraded probes or enzymes

Solutions: Verify your thermal cycler temperatures with a separate thermometer. Make sure you're mixing thoroughly. Check expiration dates on kit components.

Problem: Low RNA yield after depletion

Possible causes:

• Starting with too little RNA

• RNA degradation during the protocol

• Loss during bead purification

Solutions: Start with at least 100 ng if possible. Keep everything on ice. Be gentle when removing supernatants so you don't accidentally aspirate beads.

Problem: Residual DNA contamination

Possible causes:

• Insufficient DNase I treatment

• DNase I enzyme has lost activity

Solutions: Make sure you're incubating for the full 30 minutes at 37°C. Check that your DNase I hasn't expired.

Conclusion

Globin messenger RNA and ribosomal RNA are the background noise that drowns out the signals you care about in blood-derived samples. By removing them before cDNA synthesis, you dramatically improve the quality of your downstream data.

The depletion protocol we've covered today is thorough but straightforward. It uses DNA probes to mark unwanted RNAs, RNase H to destroy them, DNase I to clean up the DNA, and magnetic beads to purify what's left. The result is enriched messenger RNA ready for high-quality cDNA synthesis, RNA sequencing, or any other application where gene expression matters.

References

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7. Adiconis, X., et al. (2013). Comparative analysis of RNA sequencing methods. Nature Methods, 10(7), 623–629.

8. Morlan, J. D., et al. (2012). Selective depletion of rRNA enables whole transcriptome profiling. BMC Biotechnology, 12, 1–12.

9. Zhao, S., Fung-Leung, W.-P., Bittner, A., Ngo, K., & Liu, X. (2014). Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells. PLoS ONE, 9(1), e78644. https://doi.org/10.1371/journal.pone.0078644

10. Cui, P., Lin, Q., Ding, F., Xin, C., Gong, W., Zhang, L., Geng, J., Zhang, B., Yu, X., Yang, J., Hu, S., & Yu, J. (2010). A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing. Genomics, 96(5), 259–265. https://doi.org/10.1016/j.ygeno.2010.07.010

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13. New England Biolabs. (2024, June). NEBNext® globin & rRNA depletion kit (human/mouse/rat) (NEB #E7750S) (Version 4.0) [Package insert]. https://www.neb.com/en-us/products/e7750-nebnext-globin-rrna-depletion-kit-human-mouse-rat