May 11, 2026
RNA extraction from Sterivex, GFF (2.7 μm and 0.2 μm) membrane filters and cDNA library generation
- Paraskevi (VIvian) Mara1
- 1Woods Hole Oceanographic Institution
- RNA extraction and cDNA library preparation from Sterivex and GFF, 2.7 um and 0.2 um filters
Protocol Citation: Paraskevi (VIvian) Mara 2026. RNA extraction from Sterivex, GFF (2.7 μm and 0.2 μm) membrane filters and cDNA library generation. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoemoxl4o/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: May 06, 2026
Last Modified: May 11, 2026
Protocol Integer ID: 316440
Keywords: diverse marine water column microbial community, rna extraction from sterivex, rna extraction, integrated workflow for rna extraction, rna cleanup step, diverse marine water column, robust recovery of transcriptomic material, rna yield, transcriptomic material, water column sample, cdna library preparation, optimized chemical extraction step, microbial community, complexity water column sample, total rna, sequencing analysis, sequencing application, chemical extraction step, extraction procedure, cdna library generation, modified extraction procedure, dnase treatment, membrane filter
Funders Acknowledgements:
National Science Foundation
Grant ID: NSF OCE-2318228
Simons Foundation
Grant ID: 929985
National Science Foundation
Grant ID: NSF OCE-1336082
National Science Foundation
Grant ID: NSF OCE-19924492
National Science Foundation
Grant ID: NSF OCE-1259110
National Science Foundation
Grant ID: NSF OCE-1335436
Abstract
This protocol describes an integrated workflow for RNA extraction from water column samples collected on Sterivex and membrane filters (GFF, 2.7 µm, and 0.2 µm), followed by cDNA library preparation for downstream sequencing applications. It combines modified extraction procedures adapted for low-biomass and high-complexity water column samples, including in-filter lysis, bead-assisted mechanical disruption, and optimized chemical extraction steps to maximize RNA yield and integrity. The protocol further includes DNase treatment to remove residual genomic DNA, RNA cleanup steps to ensure high-quality input material, and library preparation using commercial kits optimized for total RNA and poly(A) mRNA sequencing. Together, these methods enable robust recovery of transcriptomic material from diverse marine water column microbial communities for high-throughput sequencing analyses.
Guidelines
To minimize RNase contamination, UV-sterilize the hood between steps and regularly clean pipettors and work surfaces with RNase Away or 10 M NaOH.
Ensure that heat blocks and PCR machines have reached the appropriate temperature prior to initiating reactions. Perform all reactions on ice and, when possible, use PCR coolers or chilled PCR racks/blocks to facilitate sample handling and temperature stability. PCR tubes should be placed into the thermocycler only after the target temperature has been reached.
Safety warnings
Perform the extraction directly within the filter to minimize contamination.
Ethics statement
N/A
Before start
Important: Perform all steps in UV-sterilized hoods with HEPA filtration to minimize contamination. To minimize RNase contamination, UV-sterilize the hood between steps and regularly clean pipettors and work surfaces with RNase Away or 10 M NaOH.
Ensure that heat blocks and PCR machines have reached the appropriate temperature prior to initiating reactions. Perform all reactions on ice and, when possible, use PCR coolers or chilled PCR racks/blocks to facilitate sample handling and temperature stability. PCR tubes should be placed into the thermocycler only after the target temperature has been reached.
A. RNA extraction from Sterivex filters (Qiagen kit) Note: Perform the extraction directly within the Sterivex filter to minimize contamination
Thaw Sterivex filters on ice and expel RNAlater (if used) using a 3 mL syringe. If stored at −80 °C, ensure no residual liquid remains inside the Sterivex; if present, remove it using a 3 mL syringe.
Seal the lower end (nipple) of each Sterivex filter with parafilm or Sterivex caps. Additionally, seal the lower rim with parafilm to prevent leakage.
Add beads to approximately 1/10 of the Sterivex filter volume (using beads from the 5 mL kit tubes provided by manufacturer, or UV-sterilized beads).
Aliquot 1.8 mL of PM1 buffer into 2 mL Eppendorf tubes and heat at 65 °C for 5 min.
Using a clean 3 mL syringe fitted with a 22G needle, transfer the heated PM1 buffer into each Sterivex.
Add 100 µL of molecular-grade 2-mercaptoethanol using a 1 mL syringe fitted with a 22G needle.
Seal the upper end of the Sterivex with parafilm.
Vortex at maximum speed for ~2 min.
Invert the Sterivex 180° and vortex again for ~2 min to ensure bead contact with both membrane surfaces.
Using a 3 mL syringe, pull the plunger upward to fill the syringe with air. Attach the syringe to the upper end of the Sterivex filter and apply firm pressure to recover the lysate into 2 mL tubes. Repeat this process as many times as necessary to ensure that the Sterivex is fully drained.
Centrifuge at 13,100 × g for 1 min (manufacturer instructions).
Transfer supernatant to a clean 2 mL tube and continue extraction following the manufacturer’s protocol.
Elute total RNA in 30–40 µL of RNase-free water.
B. RNA extraction from Sterivex, glass fiber (GFF), 2.7 µm and 0.2 µm filters using the mirVana™ miRNA Isolation Kit (modified from Geller-McGrath et al., 2023; https://doi.org/10.1038/s41467-023-36026-w) Note: Perform the extraction directly within the Sterivex filter to minimize contamination
Add UV-sterilized beads into UV-sterilized 15 mL Falcon tubes.
Remove GFF, 2.7 µm or 0.2 µm filters from storage using sterile pipette tips and place into RNA-cleaned, UV-sterilized glass Petri dishes.
Cut filters into small pieces using sterile scalpel blades and transfer them into Falcon tubes using either the scalpel tip or sterile 100 µL pipette tips.
For Sterivex filters, follow steps A2–A3.
Add 1.8 mL Lysis/Binding Solution (kit reagent) inside the 15 mL Falcon tubes using a pippetor (if GFF or membrane filters), or inside the Sterivex using a syringe as in step A5.
Add 100 µL β-mercaptoethanol (Sigma-Aldrich, BioUltra ≥99%), using 1 mL syringe fitted with a 22G needle.
Vortex Falcon tubes for 5 min.
For Sterivex samples, follow steps A7–A9.
Centrifuge the 15 mL Falcon tubes at 3,000 × g for 5 min (RT).
For Sterivex samples, follow steps A10–A11.
Transfer supernatant to 2 mL tubes and add 190 µL miRNA Homogenate Additive. Incubate on ice for 10 min.
Centrifuge at 13,100 × g for 1 min.
Transfer supernatant to 15 mL tubes and proceed with Acid-Phenol:Chloroform extraction (1:1 volume) per manufacturer protocol.
Centrifuge at RT for 5 min Repeat this step if phase separation is not clearly visible.
Transfer aqueous phase to new tubes and add 100% Ethanol (1.25 volumes) as per manufacturer instructions. Mix by gentle inversion to ensure homogenization.
Pass 600 µL of the lysate/ethanol mixture through the filter cartridge provided by the manufacturer. Repeat as needed until the entire sample has been processed. Complete the remaining steps according to the mirVana protocol.
Elute total RNA in 30 µL elution buffer.
C. DNase I treatment (New England Biolabs; M0303S)
Transfer 20–30 µL RNA into PCR tubes.
Add nuclease-free water (12.5–22.5 µL depending on RNA volume).
Add 5 µL DNase buffer.
Add 2.5 µL DNase I.
Adjust final volume to 50 µL.
Incubate at 37 °C for 20 min, then hold at 4 °C.
D. RNA cleanup (RNeasy MinElute Cleanup Kit; QIAGEN)
Add 50 µL RNase-free water to each 50 µL DNase-treated RNA reaction (section C).
Proceed with kit protocol.
Elute in 30 µL.
E. or proceed with RNA bead cleanup
Use 1:1 ratio of RNA beads to RNA (RNAClean XP Bead-Based Reagent; see NEBNext Ultra II protocol).
Verify absence of DNA contamination using PCR reactions with 16S primers prior to library prep.
F. cDNA library preparation
Follow Watchmaker Total RNA Library Prep Kit (Watchmaker Genomics) protocol with xGen™ Stubby Adapters (IDT), with the following modifications:
Fragmentation time: 3–6 min. Use 3 min if RNA is already partially fragmented.
Adapter concentration: 0.1 µM (from 15 µM stock), recommended for low biomass or low-water volume samples (up to 1 L).
Follow the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (poly(A) mRNA or total RNA) protocol according to the manufacturer.
Higher adaptor dilution (>100-fold in Adaptor Dilution Buffer) is recommended for low-biomass or low filtered-water-volume samples (up to 1 L).