Sep 03, 2024

Public workspaceRNA purification and cDNA synthesis

DOI
dx.doi.org/10.17504/protocols.io.ewov1nnzogr2/v1
  • 1UT Austin;
  • 2UCSF;
  • 3University of California, San Francisco
Vicki Deng
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DOI: dx.doi.org/10.17504/protocols.io.ewov1nnzogr2/v1
Protocol Citation: Vicki Deng, Fredrick leon, David Booth 2024. RNA purification and cDNA synthesis . protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1nnzogr2/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: February 04, 2022
Last Modified: September 03, 2024
Protocol Integer ID: 57845
Keywords: choanoflagellate, reverse transcription, cDNA, RNA purification
Abstract
This protocol compiles multiple methods for purifying RNA from an S. rosetta lysate and provides a modified reverse transcriptase protocol to robustly synthesize cDNA from transcripts with higher GC content.
Culture cells for lysate
Culture cells for lysate
Grow enough culture for ~50x107 cells.
  • for swimming cultures: seed Amount40 mL acclimated cells at ~Concentration8*10^4 cells/ml then culture at Temperature27 °C for Duration24:00:00
  • for thecate cultures: seed Amount100 mL acclimated cells in culture plate then culture at Temperature27 °C for Duration24:00:00

Number of cells doesn't necessarily matter. You just want enough cells to extract sufficient nucleic acid from taking in account the loss of yield in subsequent steps.





2d
Extract RNA
Extract RNA
You can extract total RNA (go to step 3.1) or mRNA (go to step 3.2)
Trizol LS total RNA extraction
  1. Add 3:1 Trizol LS to lysate and incubate for Duration00:05:00
  2. Add Amount200 µL chloroform to Amount750 µL of added Trizol LS and incubate for Duration00:03:00
  3. Centrifuged sample at Centrifigation12000 x g, 4°C, 00:15:00
  4. Collect aqueous layer

RNA precipitation
  1. Add Amount500 µL cold isopropanol per Amount750 µL of added Trizol LS to collected aqueous layer and incubate on ice for Duration00:10:00
  2. Centrifuge sample atCentrifigation12000 x g, 4°C, 00:30:00
  3. Washed pellet with cold 75% ethanol
  4. Centrifuge sample at Centrifigation7500 x g, 4°C, 00:05:00
  5. Resuspend with Concentration1 millimolar (mM) citrate ,Ph6.4 .
  6. Measure RNA concentration

or

RNeasy Cleanup
we use the RNeasy MinElute Cleanup Kit
1. Adjust the sample to a volume ofAmount100 µL
2. AddAmount250 µL 96–100% ethanol to the diluted RNA, and mix
3. Transfer the sample (Amount700 µL ) to an RNeasy MinElute spin column placed in a 2 ml collection tube. Centrifuge for Centrifigation8000 x g, Room temperature, 00:00:15 . Discard the flow-through.
4. Place the RNeasy MinElute spin column in a new 2 ml collection tube. Add Amount500 µL Buffer RPE to the spin column. Close the lid gently, and centrifuge for Centrifigation8000 x g, Room temperature, 00:00:15
5. AddAmount500 µL of 80% ethanol to the RNeasy MinElute spin column. Close the lid gently, and centrifuge for Centrifigation8000 x g, Room temperature, 00:02:00
6. Place the RNeasy MinElute spin column in a new 2 ml collection tube. Open the lid of the spin column, and centrifuge at full speed forDuration00:05:00 .
7. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube. Add Amount14 µL RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min at full speed to elute the RNA
8. For long-term storage, supplement the RNA with Concentration1 millimolar (mM) sodium citrate ,Ph6.4












1h 15m 30s
mRNA extraction
We use the NEB Magnetic mRNA Isolation Kit
  1. Equilibrate Amount100 µL beads with Amount200 µL binding buffer
  2. Add lysate and mix forDuration00:10:00
  3. Pull down beads with magnet and remove supernatant
  4. Add Amount500 µL Wash Buffer 1 and mix for Duration00:01:00
  5. Pull down beads with magnet and remove supernatant
  6. Repeat step 4-5
  7. Add Amount500 µL Wash Buffer 2 and mix for Duration00:01:00
  8. Pull down beads with magnet and remove supernatant
  9. Repeat step 7-8
  10. Add Amount500 µL Low Salt Buffer and mix for Duration00:01:00
  11. Pull down beads with magnet and remove supernatant
  12. Add Amount20 µL elution buffer (can vary elution volume for desired concentration; can vary elution buffer, we have used Nano-pure water or nylon filtered 10 mM Tris-acetate pH 8.0)
  13. Measure mRNA concentration
  14. For long-term storage, supplement the RNA with Concentration1 millimolar (mM) sodium citrate ,Ph6.4











13m
cDNA synthesis
cDNA synthesis
6m
6m
Anneal oligo d(T)20 primer to RNA sample
  1. Assemble the reaction according to this table:


AB
ComponentVolume
50 µM oligo d(T)20 primer or 2 µM gene-specific reverse primer 1 µl
10 mM dNTP mix1 µl
RNA sample (10pg-5µg total RNA or 10pg-500 ng mRNA)up to 11 µl
DEPC-treated water or nuclease-free waterto 13 µl

2. Mix and incubate reaction at Temperature65 °C for Duration00:05:00
3. Place on ice for Duration00:01:00
6m
Reverse transcription to make cDNA
  1. Vortex 5x SSIV Buffer
  2. To the annealed RNA templates from Go togo to step #4.1 , add the following components:

AB
ComponentVolume
5x SSIV Buffer4 µl
100 mM DTT1 µl
RNaseOUT™ Recombinant RNase Inhibitor1 µl
SuperScript  IV Reverse Transcriptase (200 U/μL)1 µl
4. Incubate reaction at Temperature60 °C for 10 minutes (IMPORTANT: reaction temperature is increased from kit
instructions to account for higher GC content in some transcripts)
5. Inactivate reaction by incubation at Temperature80 °C or Duration00:10:00
6. Remove RNA with incubation with Amount1 µL of RNase H at Temperature37 °C for Duration00:20:00
30m
Clone gene of interest with cDNA
Use generated cDNA in PCR reaction with gene specific primers. To verify that the RNA purfication and cDNA synthesis was successful, amplify a highly expressed transcript, such as cofillin (PTSG_01554).