Mar 02, 2026
Master Protocol: A 60-Minute Universal Green-Chemistry Workflow for RNA Extraction and Integrity Analysis
- Abdulhadi Albaser1
- 1Department of Microbiology, Faculty of Science, Sebha University, Sebha, Libya.
- Sebha university
Protocol Citation: Abdulhadi Albaser 2026. Master Protocol: A 60-Minute Universal Green-Chemistry Workflow for RNA Extraction and Integrity Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwnqx2vmk/v1
Manuscript citation:
Albaser, A. (2026). 35-minute protocol for high-yield RNA extraction from gram-positive & gram-negative bacteria at room temperature without toxic chemicals. Discover Bacteria, 3(6) DOI: https://doi.org/10.1007/s44351-025-00040-8
Albaser, A. (2026). Borax-based gel electrophoresis: A novel approach for RNA integrity analysis. PLOS ONE, 21(2), e0344092. DOI: https://doi.org/10.1371/journal.pone.0344092
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: February 28, 2026
Last Modified: March 02, 2026
Protocol Integer ID: 244214
Keywords: Green Chemistry, RNA Integrity, Borax, Formaldehyde-free, Universal Microbiology., chemistry workflow for rna extraction, rna extraction, chemistry workflow, bacteria, master protocol, minute universal green, gn, integrity analysis this protocol, yeast, gp
Disclaimer
Informational Purpose: This protocol is provided for informational and research purposes only. While the methods described have been validated in peer-reviewed publications (PLOS ONE and Discover Bacteria), users should exercise independent professional judgment when adapting these procedures to their specific laboratory settings.
Safety Warning: Although this protocol is designed to eliminate several hazardous chemicals (e.g., formaldehyde, phenol, chloroform), it involves the use of a boiling step (100°C) and electrical equipment for electrophoresis. Furthermore, if Ethidium Bromide (EtBr) is used for RNA visualization, users must handle it with extreme caution as it is a known potent mutagen and environmental hazard. Standard laboratory safety practices, including the use of personal protective equipment (PPE) such as lab coats, nitrile gloves, and eye protection, must be followed. Proper disposal of EtBr-contaminated gels and buffers according to institutional biohazardous waste guidelines is mandatory.
Liability: The author and protocols.io assume no responsibility for any injury, loss, or damage resulting from the use or misuse of the information, reagents, or steps described herein. Use of this protocol is at the risk of the individual researcher.
Abstract
This protocol is validated for Gram-Positive (GP) bacteria, Gram-Negative (GN) bacteria, and Yeast/Fungi.
Attachments
Figure1.tif
1.6MB
Image Attribution
Caption: Figure 1. Borax-based gel electrophoresis showing high-integrity 23S and 16S rRNA bands from S. aureus and E. coli.
Attribution: Image by Abdulhadi Albaser. Reproduced from Albaser (2026), PLOS ONE, 21(2): e0344092 under a Creative Commons Attribution (CC BY 4.0) License. [Link to paper: https://doi.org/10.1371/journal.pone.0344092]
Guidelines
General rules for ensuring consistent results across different microbial species.
- Sterility is Paramount: Perform all steps in an RNase-free environment. Use DEPC-treated water for all reagent preparation and keep all tubes/tips autoclaved.
- Consistency in OD: For the best yield, ensure cultures are in the mid-log growth phase (OD₆₀₀ 0.8–1.0). Overgrown cultures (stationary phase) may have lower RNA quality due to increased RNase activity.
- Temperature Control: While most of the protocol is at room temperature, the Phase I Cooling Step (Step 6) must be performed strictly on ice to ensure proper precipitation of the RNA-protein-DNA complex.
- Mixing: Always mix by gentle pipetting or inverting during the RNA isolation phase. Avoid vigorous vortexing after Step 7 to prevent shearing of the RNA.
Materials
1. Reagents & Chemicals
- For Phase I: RNA Extraction
- Acidic Lysis Buffer (pH 5.0): (Prepare using: 69.4 mM SDS, 68 mM sodium citrate dihydrate, 132 mM anhydrous citric acid, and 10 mM EDTA).
- Precipitation Buffer: 4 M NaCl with citrate/citric acid.
- Calcium Chloride (CaCl₂): 0.5 M solution (The selective RNA precipitant).
- Ethanol: 70% (Ice-cold, molecular biology grade).
- Distilled Water: Autoclaved or DEPC-treated (RNase-free).
- For Phase II: Borax Gel & Electrophoresis
- Borax (Sodium tetraborate decahydrate): Analytical grade.
- Agarose: Standard molecular biology grade.
- Nucleic Acid Stain: GelRed, SYBR Safe, or Ethidium Bromide (as per lab safety preference).
- Loading Buffer: 10X (Preferably colorless for high-resolution 5S rRNA detection).
2. Biological Materials
- Bacterial/Yeast Cultures: Mid-log phase (OD₆₀₀ 0.8–1.0). Validated for S. aureus, Enterococcus spp., E. coli, and Yeast/Fungi.
3. Equipment
- Microcentrifuge: Capable of at least 16,000 xg.
- Heat Block or Water Bath: Capable of maintaining 100°C (for boiling lysis).
- Horizontal Electrophoresis System: Tank and power supply.
- UV Transilluminator or Blue Light Imaging System.
- Vortex Mixer.
- Microwave: For melting agarose.
4. Consumables
- Microcentrifuge Tubes: 1.5 mL (Preferably "Safe-Lock" to prevent popping during the 100°C step).
- Pipette Tips: RNase-free, filter tips recommended.
- Ice Bucket and Crushed Ice.
Troubleshooting
Safety warnings
Crucial hazards and PPE requirements.
- Thermal Hazard (Boiling Step): Step 4 requires incubation at 100°C. Use a secure heat block and handle tubes with insulated gloves or forceps to avoid steam burns. Ensure tubes are "Safe-Lock" style to prevent them from popping open during heating.
- Chemical Hazard (EtBr/Stains): If using Ethidium Bromide for visualization, handle only in a designated area. EtBr is a potent mutagen. Wear double nitrile gloves and dispose of all gels and tips in specialized hazardous waste containers.
- Electrical Safety: Ensure the electrophoresis tank is properly closed and the power supply is dry before turning on the 120V current.
- UV Exposure: Use a UV shield or a closed imaging system when visualizing bands. Never look at the UV transilluminator directly with the naked eye.
Ethics statement
Formal declaration for international standards.
- Sample Source: All microbial strains used in the development of this protocol (S. aureus, Enterococcus spp., E. coli, etc.) were obtained from certified clinical isolates or standard laboratory repositories.
- Compliance: This study did not involve human subjects, vertebrate animals, or regulated higher invertebrates. All laboratory procedures were conducted in accordance with the institutional biosafety guidelines of Sebha University, Libya.
- Environmental Impact: This protocol was specifically designed as a "Green Chemistry" alternative to reduce the use of carcinogenic formaldehyde and toxic phenol in global research.
Before start
What the researcher needs to prepare before starting the 60-minute timer.
- Reagent Prep: Prepare the Acidic Lysis Buffer (pH 5.0) and 5 mM Borax Buffer in advance. Borax dissolves easily in warm water but should be cooled to room temperature before use.
- Equipment Check: Set the water bath or heat block to 100°C at least 20 minutes before starting.
- Ice: Ensure a fresh bucket of crushed ice is available for the 7-minute cooling step.
- Gel Casting: To save time, cast the Borax-Agarose gel during the 10-minute resuspension or drying phases of Phase I.
Phase I: Rapid RNA Extraction (35 Minutes)
Harvest Cells: Centrifuge 500 µL of culture (OD₆₀₀ 0.8–1.0) at 7,000 rpm for 5 mins at room temperature. Discard supernatant.
Resuspension: Resuspend the pellet in 200 µL of autoclaved distilled water.
Lysis: Add 300 µL of acidic lysis buffer (pH 5.0). Mix gently by pipetting.
Buffer Composition: 69.4 mM SDS, 68 mM sodium citrate dihydrate, 132 mM anhydrous citric acid, and 10 mM EDTA.
Cell Disruption (Boiling Step): Incubate the mixture at 100°C for 2 minutes.
Note: This step is crucial for the robust cell walls of Gram-positive bacteria and yeast.
Initial Precipitation: Add 150 µL of precipitation buffer (4 M NaCl with citrate/citric acid). Invert 10 times.
Cooling: Incubate on ice for 7 minutes, then centrifuge at 16,000 xg for 3 mins.
Selective RNA Isolation: Transfer supernatant to a new tube containing 0.5 M Calcium Chloride (CaCl₂). Incubate at room temperature for 10 minutes.
Innovation: CaCl₂ precipitates RNA while keeping DNA and proteins in solution.
Pelleting: Centrifuge at 16,000 xg for 5 mins. Discard supernatant.
Wash 6 Dry: Wash the pellet with ice-cold 70% ethanol, centrifuge for 3 mins, and air dry for 10–20 mins.
Resuspension: Dissolve the dried RNA in 50 µL of autoclaved distilled water.
Phase II: Borax-Gel Preparation (10 Minutes)
Prepare Gel: Dissolve 0.8%–1.0% agarose in 5 mM Borax solution (sodium tetraborate decahydrate).
Add Stain: Add GelRed or Ethidium Bromide to the melted agarose before casting.
Buffer: Use the same 5 mM Borax solution as the running buffer.
Phase III: Integrity Analysis (15–25 Minutes)
Borax acts as an "apparent denaturant," resolving RNA structures without pre-heating.
Sample Preparation: Mix 20 µL of RNA with 2 µL of 10X loading buffer.
Pro-Tip: Use a colorless loading buffer (no Bromophenol Blue) for the sharpest visualization of small 5S rRNA bands.
Loading: Load samples directly onto the gel. No formaldehyde pre-treatment or cooling on ice is required.
Electrophoresis: Run at 120 V for 25 minutes.
Comparison: This is significantly faster than standard TAE gels which require 60 minutes.
Visualization and Image Acquisition
Image Capture: After electrophoresis, place the gel on a UV transilluminator (302 nm or 312 nm) or a blue light imaging system.
Band Identification: For Bacteria (GP & GN): Look for two prominent, sharp bands. The upper band is the 23S rRNA (~2.9 kb) and the lower band is the 16S rRNA (~1.5 kb).
For Yeast (Eukaryotic): Look for the 28S rRNA (upper band, ~3.8–4.8 kb) and the 18S rRNA (lower band, ~1.9–2.0 kb).
Small RNA: A smaller, fainter band near the bottom of the gel represents the 5S rRNA and tRNAs.
Integrity Assessment: Ratio: In high-quality, intact RNA, the intensity of the upper band (23S/28S) should be approximately twice the intensity of the lower band (16S/18S).
Degradation: A smear of low-molecular-weight fragments or the disappearance of the 23S/28S band indicates RNA degradation.
DNA Contamination: High-molecular-weight bands near the wells indicate genomic DNA (gDNA) contamination.
Protocol references
Primary References
- Albaser, A. (2026). 35-minute protocol for high-yield RNA extraction from gram-positive & gram-negative bacteria at room temperature without toxic chemicals. Discover Bacteria, 3:6. https://doi.org/10.1007/s44351-025-00040-8
- Albaser, A. (2026). Borax-based gel electrophoresis: A novel approach for RNA integrity analysis. PLOS ONE, 21(2): e0344092. https://doi.org/10.1371/journal.pone.0344092
Foundational & Comparative References
- Brody, J. R., & Kern, S. E. (2004). Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques, 36(2): 214-216. (The foundational work for using Borax in electrophoresis).
- Rio, D. C., Ares, M. Jr, Hannon, G. J., & Nilsen, T. W. (2010). Purification of RNA. Cold Spring Harbor Protocols, 2010(6): pdb.prot4455. (The standard traditional reference you are improving upon).
- Chomczynski, P., & Sacchi, N. (2006). The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-year-old strategy that works. Nature Protocols, 1(2): 581-585. (The hazardous traditional method your protocol replaces).
- Sasagawa, Y., et al. (2013). Simple and rapid purification of plasmid DNA from Escherichia coli by calcium chloride precipitation. Journal of Microbiological Methods, 92(1): 100-102. (Supporting the use of CaCl₂ for nucleic acid precipitation).
Acknowledgements
**Procedure:**
14. Sample Preparation: Mix 20 µL of RNA with 2 µL of 10X loading buffer.
- Pro-Tip: Use a colorless loading buffer (no Bromophenol Blue) for the sharpest visualization of small 5S rRNA bands.
15. Loading: Load samples directly onto the gel. No formaldehyde pre-treatment or cooling on ice is required.**
16. Electrophoresis: Run at 120 V for 25 minutes.
- Comparison: This is significantly faster than standard TAE gels which require 60 minutes.
17. Visualization and Image Acquisition**
**Time: 5 Minutes
**Procedure:**
1. Image Capture: After electrophoresis, place the gel on a UV transilluminator (302 nm or 312 nm) or a blue light imaging system.
2. Band Identification: * For Bacteria (GP & GN): Look for two prominent, sharp bands. The upper band is the 23S rRNA (~2.9 kb) and the lower band is the 16S rRNA (~1.5 kb).
- For Yeast (Eukaryotic): Look for the 28S rRNA (upper band, ~3.8–4.8 kb) and the 18S rRNA (lower band, ~1.9–2.0 kb).
- Small RNA: A smaller, fainter band near the bottom of the gel represents the 5S rRNA and tRNAs.
3. Integrity Assessment: * Ratio: In high-quality, intact RNA, the intensity of the upper band (23S/28S) should be approximately twice the intensity of the lower band (16S/18S).
- Degradation: A smear of low-molecular-weight fragments or the disappearance of the 23S/28S band indicates RNA degradation.
- DNA Contamination: High-molecular-weight bands near the wells indicate genomic DNA (gDNA) contamination.
**Why this protocol is unique for your applications:**
- Accessibility: It works at room temperature with minimal equipment.
- Safety: It is 100% free of phenol, chloroform, and formaldehyde.
- Versatility: It is a truly universal method for diverse microbial taxa.