Jan 10, 2026
RNA Isolation Using QIAzol-RNeasy Mini Kit and cDNA Synthesis
- Ali Ghoochani1,2,3,4,
- Monther Abu-Remaileh1,2,3,4
- 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
- 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
- 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- asap
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. RNA Isolation Using QIAzol-RNeasy Mini Kit and cDNA Synthesis . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5k496v1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 24, 2025
Last Modified: January 10, 2026
Protocol Integer ID: 233377
Keywords: rna isolation, reliable cdna generation for downstream gene, capacity cdna reverse transcription kit, reproducible rna recovery, cdna synthesis, rna before reverse transcription, cdna synthesis with the high, quality rna, rna, reliable cdna generation, reverse transcription, using qiazol, qiazol lysi, downstream gene, rneasy mini kit, rneasy mini column, column purification
Abstract
This protocol outlines a streamlined workflow for isolating high-quality RNA using QIAzol and RNeasy Mini columns, followed by cDNA synthesis with the High-Capacity cDNA Reverse Transcription Kit. It covers cell harvesting, QIAzol lysis, phase separation, column purification, and RNA before reverse transcription. The method provides reproducible RNA recovery and reliable cDNA generation for downstream gene-expression applications.
Materials
Materials & Reagents
- QIAzol Lysis Reagent
- RNeasy Mini Kit (spin columns, buffers RWT, RPE, collection tubes)
- Chloroform
- 100% ethanol
- PBS (RNase-free)
- Trypsin (0.1–0.25%)
- High-Capacity cDNA Reverse Transcription Kit (ThermoFisher)
- RNase-free water
- RNase-free tubes and pipette tips
Equipment
- Centrifuge capable of 12,000 × g
- Microcentrifuge
- 37°C block or thermal cycler
- Vortex mixer
- RNase-free hood recommended
Troubleshooting
PART I: RNA ISOLATION (QIAzol-RNeasy Mini Kit)
Suspension Cells (≤1 × 10⁷ cells)
- Count the cells.
- Pellet cells at 300 × g for 5 min.
- Carefully aspirate all supernatant
Note
Remaining medium inhibits lysis and reduces RNA yield.
Adherent Cells (≤1 × 10⁷ cells)
Trypsinize and pellet
- Count cells.
- Aspirate medium, wash once with PBS.
- Add 0.1–0.25% trypsin until cells detach.
- Add medium (with serum) to neutralize trypsin.
- Transfer to RNase-free tube and spin 300 × g for 5 min.
- Aspirate supernatant completely.
Note
Medium carryover dilutes QIAzol and lowers RNA yield.
For pelleted cells:
- Loosen pellet by flicking.
- Add 700 μL QIAzol Lysis Reagent.
- Vortex or pipette to mix thoroughly.
For direct lysis in dish:
- Add 700 μL QIAzol directly to dish.
- Scrape using a cell scraper.
- Transfer lysate to RNase-free tube and vortex to remove clumps.
Add 140 μL chloroform.
Cap and shake vigorously 15 sec.
Incubate 2–3 min at RT.
Centrifuge 15 min at 12,000 × g, 4°C.
Phases after spin: Upper aqueous phase (~350 μL): RNA
Transfer the aqueous phase to a new tube.
Add 1.5 volumes 100% ethanol (≈525 μL).
Mix well by pipetting (precipitate may form).
Load ≤700 μL into RNeasy spin column.
Spin 15 sec at 8000 × g.
Discard flow-through and reuse collection tube.
Repeat until entire sample is loaded.
Add 700 μL Buffer RWT, spin 15 sec.
Add 500 μL Buffer RPE, spin 15 sec.
Add 500 μL Buffer RPE, spin 2 min to dry membrane.
Note
Important: Avoid column contact with flow-through to prevent ethanol carryover.
Transfer column to new tube and spin 1 min full speed to remove residual buffer.
Transfer column to 1.5 mL RNase-free tube.
Add 30–50 μL RNase-free water directly onto membrane.
Spin 1 min at 8000 × g.
For high yield (>30 μg), repeat elution.
For higher concentration, elute second time using first eluate (15–30% lower final yield).
PART II: cDNA SYNTHESIS (High-Capacity cDNA Kit)
Prepare 2X RT Master Mix (per 20 μL reaction)
| Component | With RNase Inhibitor | Without RNase Inhibitor | |
| 10X RT Buffer | 2.0 μL | 2.0 μL | |
| 25X dNTP Mix (100 mM) | 0.8 μL | 0.8 μL | |
| 10X RT Random Primers | 2.0 μL | 2.0 μL | |
| MultiScribe Reverse Transcriptase | 2.0 μL | 2.0 μL | |
| RNase Inhibitor | 1.0 μL | - | |
| Nuclease-free water | 3.2 μL | 4.2 μL | |
| Total | 10 μL | 10 μL |
Prepare on ice.
Pipette 10 μL of 2X RT master mix into each tube.
Dilute the RNA to the desired input amount (e.g., 0.1–1.0 µg total RNA) in a final volume of 10 µL using RNase-free water.
Mix gently and keep on ice until adding to the 2X RT master mix.
Quick spin to collect contents and remove air bubbles.
Keep on ice until loading thermal cycler.
Set reaction volume: 20 μL
| Step | Temperature | Time | |
| 1 | 25°C | 10 min | |
| 2 | 37°C | 120 min | |
| 3 | 85°C | 5 min | |
| 4 | 4°C | Hold |
Start the run.