Direct-zol Miniprep workflow for RNAlater samples
After more than 20 years at the bench, I’ve extracted RNA using just about every approach imaginable. I’ve worked with TRIzol, as well as column-based kits from manufacturers such as Qiagen and Invitrogen’s PureLink range. Each method has its merits, but if there’s one thing I’ve learnt, it’s that RNA extraction is as much about choosing your battles as it is about getting a good yield.
In my experience, TRIzol has often delivered the highest RNA yields. The catch? Chloroform. It’s an effective part of the extraction process, but it brings its own set of headaches: working with a hazardous solvent, handling the waste, and making sure everything is disposed of properly. The RNA might be precious, but the waste certainly isn’t something you want accumulating in the corner of the lab!
Column-based kits offer a cleaner, more straightforward workflow, although yields can sometimes be lower than with TRIzol, depending on the sample and extraction conditions.
So, where does that leave us? Somewhere between the old-school chemistry of TRIzol and the convenience of spin columns lies the Direct-zol RNA MiniPrep Kit from Zymo Research.
It offers an appealing middle ground by combining TRIzol-type sample lysis with column-based RNA purification, without requiring the usual chloroform extraction step.
You'll need
Kit
Zymo Research Direct-zol RNA Miniprep
R2050 — 50 preps, no TRI Reagent
R2051 — 50 preps, includes 50 mL TRI Reagent
R2052 — 200 preps, no TRI Reagent
R2053 — 200 preps, includes 200 mL TRI Reagent.
If you don't already have TRI Reagent For your experiment, R2051 is the convenient choice.
You'll also need:
RNase-free microcentrifuge tubes
RNase-free pipette tips
microcentrifuge capable of 10,000–16,000 × g
vortex
95–100% ethanol
appropriate PPE/fume hood for handling TRI Reagent
Part 1 — Prepare the buffers
Do this before starting the samples.
Direct-zol RNA PreWash
Add:
10 mL 95–100% ethanol to the 40 mL PreWash concentrate.
Mix thoroughly.
RNA Wash Buffer
Add:
48 mL 100% ethanol to the 12 mL RNA Wash Buffer concentrate.
Alternatively, the manufacturer's protocol permits 52 mL of 95% ethanol.
DNase I
The kit's lyophilised DNase I is reconstituted with:
275 µL DNase/RNase-free water
for the 1,500-U preparation.
Mix gently by inversion rather than vigorous vortexing and prepare frozen aliquots according to the manufacturer's instructions.
Part 2 — Deal with the RNAlater sample
Step 1 — Recover the cellular pellet
The RNAlater aliquots contain preserved parasite/RBC pellet.
a) Centrifuge the sample at up to 5,000 × g to pellet the cellular material.
Zymo recommends removing the RNAlater supernatant before proceeding.
Step 2 — Remove RNAlater
b) Carefully aspirate the RNAlater supernatant.
Try to remove as much as possible without disturbing the pellet.
Step 3 — Do not wash unnecessarily
You do not need to automatically introduce a PBS wash at this stage. Every additional wash/pellet is an opportunity to lose material, which is exactly what we don't want.
Zymo's recommended RNAlater approach is simply:
pellet → remove RNAlater → lyse the pellet in TRI Reagent.
Part 3 — TRI Reagent lysis
With the TRI reagent, we're effectively using the liquid extraction chemistry, but without the phenol/chloroform phase separation.
Step 4 — Add TRI Reagent
Resuspend the pellet thoroughly in an appropriate volume of TRI Reagent.
The Zymo protocol's cell guidance uses approximately:
≤10⁵ cells → ≥100 µL TRI Reagent
≤10⁶ cells → ≥300 µL
≤5 × 10⁶ cells → ≥600 µL.
Do not blindly apply the cell-number table; the practical objective is to completely lyse and homogenise the pellet. Use a needle and syringe to suction the cells up-and-down, if needed.
If the lysate remains very viscous or contains obvious particulate material, Zymo recommends increasing the TRI Reagent volume until the lysate is properly homogenised.
Step 5 — Homogenise thoroughly
Vortex/mix until the pellet is completely disrupted.
You don't want visible clumps before proceeding. This is especially important because incomplete lysis is one of the manufacturer's identified causes of poor recovery/viscous lysates.
Part 4 — Direct-zol binding
This is where the workflow becomes much nicer than conventional TRIzol extraction.
Step 6 — Add an equal volume of ethanol
Take your homogenised sample in TRI Reagent.
Add: 1 volume 95–100% ethanol to: 1 volume TRI Reagent lysate
Example:
400 µL lysate + 400 µL ethanol
Mix thoroughly.
You do not need to perform chloroform extraction. That's the key advantage of Direct-zol. The manufacturer specifically instructs you to add ethanol directly to the TRI Reagent lysate and load it onto the column.
Part 5 — Load the Zymo column
Step 7 — Load
Transfer the lysate/ethanol mixture to a Zymo-Spin IICR Column in its collection tube.
Centrifuge at: 10,000–16,000 × g for 30 seconds according to the current protocol.
If your sample exceeds the column's loading volume, load it in successive portions.
The manufacturer specifically notes that samples >700 µL need to be reloaded and centrifuged again.
Transfer the column to a fresh collection tube. Discard the flow-through.
Part 6 — DNase treatment
Step 8 — First wash
Add: 400 µL Direct-zol RNA Wash Buffer
to the column.
Centrifuge.
Discard flow-through.
Step 9 — DNase
Prepare:
5 µL DNase I
75 µL DNA Digestion Buffer
Prepare a mastermix depending on the no. of samples you are extracting from.
Example
Mix gently.
Apply the entire 80 µL mixture directly to the centre of the column membrane.
Step 10 — Incubate
Incubate: 15 minutes at room temperature (20–30°C).
This is the manufacturer's recommended on-column DNase treatment.
Part 7 — Wash
Step 11 — PreWash
Add: 400 µL Direct-zol RNA PreWash
Centrifuge.
Discard flow-through.
Repeat:
400 µL PreWash
Centrifuge again.
Step 12 — RNA Wash Buffer
Add: 700 µL RNA Wash Buffer
Centrifuge for: 1 minute at 10,000–16,000 × g.
This centrifugation ensures removal of residual wash buffer.
Part 8 — Elute
Step 13 — Transfer column
Move the column into a clean RNase-free tube.
Step 14 — Elute
Add: 50 µL DNase/RNase-free water directly to the centre of the membrane.
Centrifuge.
That gives you your RNA.
Zymo permits an elution volume of ≥25 µL if you want a more concentrated preparation.
It’s a good idea to use 30–50 µL initially.
If you have samples that contain very little RNA, go for recovery over maximum concentration unless the RNA yield proves to be extremely low.
You can always concentrate RNA later.
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