Sep 01, 2020

Public workspacePurification of RNA from a DNA/RNA Extract V.2

DOI
dx.doi.org/10.17504/protocols.io.bftbjnin
  • 1Soil and Water Research Infrastructure
  • Anaerobic and Molecular Microbiology Lab, Biology Centre CAS
    Tech. support email: eva.petrova@bc.cas.cz
Roey Angel
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bftbjnin
Protocol Citation: Roey Angel, Eva Petrova 2020. Purification of RNA from a DNA/RNA Extract. protocols.io https://dx.doi.org/10.17504/protocols.io.bftbjnin
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: April 30, 2020
Last Modified: September 01, 2020
Protocol Integer ID: 36419
Keywords: RNA, DNase, nucleic acid purification,
Abstract
The following protocol is intended as a downstream application for our Total Nucleic Acids Extraction from Soil protocol. This protocol describes how to purify RNA from a DNA and RNA extract using TURBO™ DNase and GeneJET RNA Cleanup and Concentration Micro Kit. This protocol is a simplified and condensed version of the full protocols provided by the manufacturers.
Attachments
Icon for 4393900B.pdf
4393900B.pdf
281KB
Icon for MAN0012671_GeneJET_RNA_Cleanup_Concentration_Micro_UG.pdf
MAN0012671_GeneJET_R...
276KB
Icon for THE RNA storage solution.pdf
THE RNA storage solu...
205KB
Materials
MATERIALS
ReagentTURBO™ DNase (2 U/µL)Thermo Fisher ScientificCatalog #AM2238
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentEthanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
ReagentRNase AWAY™ Surface DecontaminantCarl RothCatalog #A998.4
ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
ReagentRNaseOUT Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
STEP MATERIALS
ReagentTURBO™ DNase (2 U/µL)Thermo Fisher ScientificCatalog #AM2238
ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
ReagentUSB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
ReagentNuclease-free autoclaved DEPC-treated waterCarl RothCatalog #T143.1
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentEthanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
Protocol materials
ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
ReagentUSB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
ReagentTURBO™ DNase (2 U/µL)Thermo Fisher ScientificCatalog #AM2238
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
ReagentEthanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
ReagentRNase AWAY™ Surface DecontaminantCarl RothCatalog #A998.4
ReagentTURBO™ DNase (2 U/µL)Thermo Fisher ScientificCatalog #AM2238
ReagentEthanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
ReagentRNaseOUT Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
ReagentNuclease-free autoclaved DEPC-treated waterCarl RothCatalog #T143.1
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentTURBO™ DNase (2 U/µL)Thermo Fisher ScientificCatalog #AM2238
ReagentUSB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
ReagentNuclease-free autoclaved DEPC-treated waterCarl RothCatalog #T143.1
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentEthanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
Before start
1. For each sample prepare one Gene JET RNA Purification Micro Column tube and two RNase–free collection tubes (1.5 ml).
2. Add the required amount of ethanol to Wash Buffer 1 and Wash Buffer 2 (amount is dependent on kit size).
DNA digestion
DNA digestion
45m
45m
Prepare the following mixture in a 1.5 ml tube:
  1. Amount10 µL to Amount42 µL of TNA extract (Amount1 µg to Amount3 µg of DNA).
  2. Amount5 µL TURBO DNase buffer 10x
  3. Amount1 µL RNaseOUT
  4. Amount1 µL 0,1M DTT
  5. Amount1 µL Turbo DNase per up to Amount2 µg DNA
  6. Complete to Amount50 µL with RNase-free water
ReagentTURBO™ DNase (2 U/µL)Thermo Fisher ScientificCatalog #AM2238
ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019


ReagentUSB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML

ReagentNuclease-free autoclaved DEPC-treated waterCarl RothCatalog #T143.1

Pipetting
Incubate at Temperature37 °C for Duration00:30:00 .

30m
Digestion
Step case

Extended DNA digestion
10 steps

If this procedure still leaves out undigested DNA (for example due to the presence of inhibitors), increase the incubation time (to 40–60 min) and add another equal dose of DNase half-way through.
RNA purification
RNA purification
7m
7m
Add Amount250 µL Binding Buffer .

ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
Pipetting
Add Amount300 µL absolute ethanol .

ReagentEthanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
Pipetting
Transfer the mixture to the Gene JET RNA Purification Micro Column preassembled with a collection tube. Centrifuge the column for Centrifigation14000 x g, Room temperature, 00:01:00 . Discard the flow-through. Place the GeneJET RNA Purification Micro Column back into the collection tube.

1m
Centrifigation
Add Amount700 µL Wash Buffer 1 (supplemented with ethanol) to the GeneJET RNA Purification Micro Column and centrifuge for Centrifigation14000 x g, Room temperature, 00:01:00 . Discard the flow-through and place the purification column back into the collection tube.
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
1m
Wash
Add Amount700 µL Wash Buffer 2 (supplemented with ethanol) to the GeneJET RNA Purification Micro Column and centrifuge for Centrifigation14000 x g, Room temperature, 00:01:00 . Discard the flow-through and place the purification column back into the collection tube.
ReagentGeneJET RNA Cleanup and Concentration Micro KitThermo Fisher ScientificCatalog #K0841
1m
Wash
Repeat step 7.
Go togo to step #7
1m
Wash
Centrifuge the empty GeneJET RNA Purification Micro Column for an additional Centrifigation14000 x g, Room temperature, 00:02:00 to completely remove residual Wash Buffer.
Note
This step is essential to avoid residual ethanol in the purified RNA solution. The presence of ethanol in the RNA sample may inhibit downstream enzymatic reactions.

2m
Centrifigation
Transfer the GeneJET RNA Purification Micro Column into a clean 1.5 ml Collection Tube tube.


Add Amount10 µL to Amount20 µL RNA storage solution or nuclease-free water to the GeneJET RNA Purification Micro Column. Centrifuge for Centrifigation14000 rpm, Room temperature, 00:01:00 to elute the RNA.
ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000

1m
Centrifigation
Discard the purification column. Use the purified RNA immediately in downstream applications or store at Temperature-20 °C or Temperature-80 °C until use. Proceed to cDNA synthesis using superscript IV

Note
For prolonged storage (more than 1 month), storage at Temperature-80 °C is recommended.


Protocol
cDNA synthesis using SuperScript™ IV
NAME
cDNA synthesis using SuperScript™ IV
CREATED BY
Roey Angel

Prepare the following mixture in a PCR tube:

  1. Amount1 µL to Amount4 µL purified RNA (Amount10 pg - Amount5 µg ; usually Amount200 ng for soil extract)
  2. Amount1 µL random hexamers (50 µM) or a gene-specific primer (Amount2 µL )
  3. Amount9.8 µL RNase free water

ReagentRandom hexamersThermo ScientificCatalog #N8080127

ReagentNuclease-free autoclaved DEPC-treated waterCarl RothCatalog #T143.1

Pipetting
Mix gently and spin down the solution.
Mix
Incubate the mixture at Temperature65 °C for Duration00:05:00 in a thermocycler and chill TemperatureOn ice (or in the cycler at >Temperature4 °C ) for at least Duration00:01:00 .
Incubation
Prepare the following mixture and add to each tube:

  1. Amount4 µL 5x Reaction buffer
  2. Amount1 µL dNTP mix, 10 mM
  3. Amount1 µL 0.1M DTT
  4. Amount1 µL RNaseOUT™ (40 U/µl) * 
  5. Amount0.2 µL BSA (20 µg/µl)
  6. Amount1 µL SuperScript™ IV RT (200 units/μl)
*  Optional

ReagentUSB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML

ReagentSuperScript™ IV First-Strand Synthesis SystemThermo Fisher ScientificCatalog #18091050
ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
ReagentBovine Serum Albumin (BSA)Thermo Fisher ScientificCatalog #B14
Pipetting
Incubate the mixture in a thermocycler atTemperature23 °C for Duration00:10:00 (only if using random hexamers, skip if using a specific primer) followed by Temperature50 °C for Duration01:00:00 to Duration03:00:00 and Temperature80 °C for Duration00:10:00 . Chill TemperatureOn ice .
Incubation
For PCR templates > 1kb remove the RNA by adding Amount1 µL (2 units) of E. coli RNase H and incubate atTemperature37 °C for Duration00:20:00 .
ReagentRibonuclease H (RNase H)Thermo Fisher ScientificCatalog #18021071

Optional
Prepare the following mixture and add to each tube:

  1. Amount1 µL DNA Polymerase I reaction buffer    
  2. Amount0.75 µL DNA Polymerase I
  3.  Amount0.2 µL RNase H              
  4. Amount3.05 µL RNase-free water                                
  5. Amount5 µL Template cDNA
ReagentDNA Polymerase I (10 U/µL)Thermo Fisher ScientificCatalog #EP0041
ReagentRibonuclease H (RNase H)Thermo Fisher ScientificCatalog #18021071

ReagentNuclease-free autoclaved DEPC-treated waterCarl RothCatalog #T143.1

Optional
Incubate for at Temperature15 °C for Duration02:00:00 followed by Duration00:10:00 at Temperature75 °C for deactivation.

Incubation
3. Purify the reaction through phenol/chloroform purification followed by ethanol precipitation or using a PCR purification kit.