Jan 24, 2020

Public workspaceHigh-quality RNA purification with on-column DNase treatment from tissue specimens

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.8ufhwtn
  • 1National and Kapodistrian University of Athens;
  • 2Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven – University of Leuven, Leuven, Belgium;
  • 3Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA;
  • 4Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin Humbolt-Universität zu Berlin and Berlin Institute of Health, Institute of Virology, Berlin, Germany
  • PLOS ONE Lab Protocols
    Tech. support email: plosone@plos.org
Magda Bletsa
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DOI: dx.doi.org/10.17504/protocols.io.8ufhwtn
External link: https://doi.org/10.1371/journal.pone.0324537
Protocol Citation: Magda Bletsa, Antonios Fikatas, Sophie Gryseels, Jan Felix Drexler, Philippe Lemey, Yiqiao Li 2020. High-quality RNA purification with on-column DNase treatment from tissue specimens . protocols.io https://dx.doi.org/10.17504/protocols.io.8ufhwtn
Manuscript citation:
Li Y, Polychronopoulou M, Boonen I, Fikatas A, Gryseels S, Laudisoit A, Bellocq JGd, Vrancken B, Magiorkinis G, Lemey P, Bletsa M (2025) Evaluation of metatranscriptomic sequencing protocols to obtain full-length RNA virus genomes from mammalian tissues. PLOS One 20(5). doi: 10.1371/journal.pone.0324537
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
This protocol can be used for total RNA purification from tissue samples. It includes an intermediate on-column DNase treatment, which results in higher RNA yield and purity. This method is mainly optimised to obtain hepacivirus RNA extracts for whole-genome sequencing, but it can also be used for any viral RNA purification from samples with lower viral loads.
Created: October 29, 2019
Last Modified: January 24, 2020
Protocol Integer ID: 29287
Keywords: RNA extraction, hepacivirus, tissue
Abstract
This protocol can be used for total RNA purification from tissue samples. It includes an intermediate on-column DNase treatment, which results in higher RNA yield and purity. This method is mainly optimised to obtain hepacivirus RNA extracts for whole-genome sequencing, but it can also be used for any viral RNA purification from samples with lower viral loads.
Guidelines
  • During tissue handling, all procedures should be carried out as quickly as possible.
  • Only RNA molecules > 200 nucleotides are purified.
  • Do not overload the RNeasy spin column (maximum capacity of Amount700 µL ).
  • Buffer RLT may form a precipitate upon storage. Re-dissolve by putting the bottle under warm water for a few minutes.
  • Perform all steps at TemperatureRoom temperature , unless otherwise stated.
  • Always use new collection tubes to eliminate any possible contamination.
Materials
MATERIALS
ReagentRNeasy Mini KitQiagenCatalog #74104
ReagentRNase-Free DNase SetQiagenCatalog #79254
ReagentPrecellys CK28 Lysing Kit Hard Tissue Homogenizing BertinVWR International (Avantor)Catalog #10144-516
STEP MATERIALS
ReagentPrecellys CK28 Lysing Kit Hard Tissue Homogenizing BertinVWR International (Avantor)Catalog #10144-516
Protocol materials
ReagentRNeasy Mini KitQiagenCatalog #74104
ReagentRNase-Free DNase SetQiagenCatalog #79254
ReagentPrecellys CK28 Lysing Kit Hard Tissue Homogenizing BertinVWR International (Avantor)Catalog #10144-516
ReagentPrecellys CK28 Lysing Kit Hard Tissue Homogenizing BertinVWR International (Avantor)Catalog #10144-516
ReagentPrecellys CK28 Lysing Kit Hard Tissue Homogenizing BertinVWR International (Avantor)Catalog #10144-516
Before start
  • When using Buffer RPE for the first time, add 4 volumes of ethanol (100%).
  • Prepare DNase I stock solution of RNase-free DNase set as described: Do not open the glass vial. Transfer Amount550 µL of the RNase-free water provided into a Amount1.5 mL Eppendorf tube. Using a needle and a syringe inject the water into the lyophilised DNase I glass vial. Mix gently by inverting the vial and make sure the powder on the sides of the vial is all well dissolved. DO NOT VORTEX. For long-term storage of DNase I, remove the stock solution from the glass vial, using the syringe. Divide it into Amount100 µL aliquots and store at --Temperature20 °C ℃ for up to 9 months. Thawed aliquots can be stored at Temperature4 °C for up to 6 weeks. Do not refreeze the aliquots after thawing.








Sample homogenisation
Sample homogenisation
Add Amount600 µL RLT Buffer to the Precellys lysate tubes.
ReagentPrecellys CK28 Lysing Kit Hard Tissue Homogenizing BertinVWR International (Avantor)Catalog #10144-516


Excise a lentil-sized piece of tissue (maximum amount of Amount20 mg for RNAlater stabilized tissues and Amount30 mg for fresh or frozen tissues) and transfer it quickly to the lysate tubes. Make sure that all tissues are immersed into the RLT reagent.

Place tubes on dry ice for Duration00:02:00 (or until frozen) and then thaw quickly.

Immediately after thawing disrupt the tissues using a conventional rotor-stator homogeniser (Minilys). The recommended lysis should be performed at medium speed (Centrifigation4000 rpm ) for Duration00:02:00 .
Equipment
Minilys Personal Homogeniser
NAME
Tissue homogeniser
TYPE
Bertin Instruments
BRAND
P000673-MLYS0-A
SKU
https://www.bertin-instruments.com/product/sample-preparation-homogenizers/minilys-tissue-homogenizer/
LINK




Place tubes on dry ice for Duration00:02:00 (or until frozen) and then thaw quickly.
Check the results and repeat the homogenisation until no more visible fragments are present.
Centrifuge the lysate for Duration00:03:00 at full speed. Carefully remove the supernatant by pipetting, and transfer it to a new Amount1.5 mL Eppendorf tube.

RNA binding
RNA binding
Add Amount600 µL 70% ethanol to the supernatant, and mix immediately by pipetting 5 times. DO NOT VORTEX OR CENTRIFUGE. Proceed immediately to the next step.

TransferAmount700 µL of the sample to an RNeasy spin column placed in a Amount2 mL collection tube. Centrifuge for Duration00:00:30 at Centrifigation10000 rpm
If the sample is more than Amount700 µL , change collection tubes and transfer the rest to the spin column and centrifuge again.
Discard the collection tubes and replace with new ones.
AddAmount350 µL Buffer RW1 to the RNeasy spin column and centrifuge for Duration00:00:30 at Centrifigation10000 rpm to wash the spin column membrane. Change collection tubes.


DNase treatment
DNase treatment
Prepare the DNase treatment master mix, as described:
For each sample, add Amount10 µL DNase I stock solution to Amount70 µL Buffer RDD. Mix by gently inverting the tube, and spin down briefly. DO NOT VORTEX.

Add the Amount80 µL DNase I incubation mix directly to the RNeasy spin column membrane, and place on benchtop atTemperatureRoom temperature for Duration00:15:00 .

Washing steps
Washing steps
AddAmount350 µL Buffer RW1 to the RNeasy spin column and centrifuge for Duration00:00:30 at Centrifigation10000 rpm to wash the spin column membrane. Change collection tubes.

AddAmount500 µL Buffer RPE to the RNeasy spin column and centrifuge for Duration00:00:30 at Centrifigation10000 rpm . Change collection tubes.

AddAmount500 µL Buffer RPE to the RNeasy spin column and centrifuge for Duration00:02:00 at Centrifigation10000 rpm . Change collection tubes.

Dry centrifugation
Dry centrifugation
Place the RNeasy spin column in a new collection tube and centrifuge at full speed for Duration00:05:00 If there is still much liquid passing through the column, change collection tubes and centrifuge for Duration00:01:00 .

Elution and storage
Elution and storage
Place the spin column in a new Amount1.5 mL Eppendorf tube. Add Amount50 µL RNase-free water directly to the spin column membrane. Incubate for Duration00:05:00 at TemperatureRoom temperature . Then centrifuge for Duration00:01:00 at Centrifigation10000 rpm to elute the RNA.

Repeat step 16. Use a new Amount1.5 mL tube for another elution round of Amount50 µL .

Store at -- Temperature20 °C . Optionally, you can put the column back to the corresponding collection tube and store them at -- Temperature20 °C for another future elution. Even after the 3rd or 4th elution time, there are still some RNA molecules passing through the column.