Ds cDNA synthesis

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NB! pre-PCR operation
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    [править] First-strand synthesis

    total amount of EB is wrong calculated

  1. calculate recipe for each RNA sample:

    number of samples:
    RNAH2O3.3x FS-mixRPoligo(dT)FS
    enz. mix

  2. * RP: random primers, 50ng/µl
    * oligo(dT): oligo(dT) primer, 50 µM

  3. prepare fresh 3.3x FS mix (First Strand synthesis mix) in a small excess[1]
    Reverse transcription buffer, 10x3.33x1µl33.3µl
    dNTP's, 10mM1.67mM0.5µl16.7µl
    MgCl2, 0.1M16.7mM0.5µl16.7µl
    DTT, 0.1M33mM1µl33.3µl

  4. mix in individual(!) 0.2ml PCR tubes:
    • in case of non shared RNA everything except for the "enzyme mix" and EB;
    • in case of shared RNA RNA and random primers (concentration of random primers should be 3µg/µl)

  5. PCR-machine[2][3]:
    Non sheared RNASheared RNA

    98°C 1:00
    70°C 5:00
    0,1°C to 15°C
    15°C 30:00
    15°C hold

    65°C 5:00
    15°C hold
    In PCR machine add and mix 3.3x FS mix
    Wait about 2 min and go to the next step

  6. in PCR-machine: add and mix carefully FS enzyme mix (for sheared RNA with amount up to 300ng use 200units (1µl) of SSIII )[4];
  7. go to the next step of the PCR-program[5]:
    1. 0.1°C to 25°C
    2. 25°C 10:00
    3. 0.1°C to 42°C
    4. 42°C 45:00
    5. 0.1°C to 50°C
    6. 50°C 15:00
    7. 75°C 15:00
    8. 15°C hold

    add   EB   to 30µl

    [править] Second-strand synthesis

    • prepare in advance:
    » 16°C waterbath
    » commercial G-50 micro spin columns[6]
    » autoclaved G-50 suspension
    » tips with GF-C filters for preparation of gel-filtration columns

  8. purify reaction using gel-filtration spin-column: G-50, 1mM TrisCl, pH 7.0
    • spin empty column: 0.5min at 2krpm;
    • insert column into new signed collection tube;
    • load sample and spin: 1min at 2krpm;
  9. measure volume of eluate and transfer it into new 1.7ml tube;
  10. put tube on ice;
  11. calculate recipe for each RNA sample:
    RNAH2O3.3x SS-mixQG bufferIsopropanoltotal

  12. prepare fresh 3.3x SS-mix (Second Strand synthesis mix) mix in a small excess[7]
    conc.1 rxn5.33 rxn
    Reverse transcription buffer, 10x0.44x1µl5.3µl
    Second str. synth. buffer, 5x3.33x15µl80µl
    MgCl2, 0.1M2.2mM0.5µl2.7µl
    DTT, 0.1M4.4mM1µl5.3µl
    dUNTP's, 10mM0.89mM2µl10.7µl
    E.coli DNA ligase, 10u/µl0.22u/µl0.5µl2.7µl
    DNA pol. I, 10u/µl0.89u/µl2µl10.7µl
    RNase H, 2u/µl44u/ml0.5µl2.7µl
    total: 22.5µl120µl

  13. add H2O and 3.33x SS-mix, mix carefully;
  14. 16°C, 2 hours;
  15. QIAquick-QG purification   (3x QG, 1x Iso),   elute with 50µl EB (2x26µl)[8];
  16. determine DNA concentration on Qubit (DNA HS kit);
  17. snap freeze, store at -20°C

[править] Solutions

FS enzyme mix First Strand enzyme mix, prepare beforehand and store at -20°C

RNase OUT, 40u/µl20u/µl5µl100µl
SuperScript III, 200u/µl100u/µl5µl100µl

dUNTP's, 10mM store at -20°C

dATP, 100mM10mM5µl10µl
dCTP, 100mM10mM5µl10µl
dGTP, 100mM10mM5µl10µl
dUTP, 100mM10mM5µl10µl

[править] Notes

  • typically, ~0.2-0.3µg of cDNA is synthesised from 0.3µg of mRNA.

  • if RNA is sheared beforehand, oligo(dT) primer is not included into First-strand synthesis reaction. In this case ds cDNA is ready for library construction without shearing.

  • if "long" RNA is used as a starting material, ds cDNA should be sheared before sequencing library construction.

  • It is possible to stop after:
» first-strand synthesis
» spin-column purification
» second-strand synthesis (snap-freeze w/o purification)

  • First-strand synthesis:
» total RNA requires 1/4 of primers and enzymes if compare with polyA+ RNA;
» reaction may be performed in 10µl volume for up to 0.5µg of mRNA;
» maximum First-strand synthesis volume for different types of gel-filtration columns
columnmax. sample volume
1ml (blue tip)100µl
200µl (yellow tip)30µl
ProbeQuant G-5050µl

  • Substitution of dTTP by dUTP during second-strand synthesis:
» practically does not change DNA synthesis rate;
» even 75% substitution totally prevents "reverse reads" during synthesis;
» Uridine-containing strand should be destroyed before library amplification by UDGase.

Open questions

  • Actinomycin D prevents formation of double-stranded cDNA during First-strand synthesis. It has practically no effect if ds cDNA is prepared from "long mRNA". It is worth to check influence of Actinomycin D on cDNA synthesis from "short" sheared mRNA.

  • Actinomycin D, Mw 1255g/mol: Sigma #A1410, 2mg;

  • Stock solution: 1M
» add of 1ml of H2O mQ to 2mg of Actinomycin D
» check concentrtion on spectrophotometer (extinction coefficient at 441nm: 21900 L/mol*cm)
solution: {97.8µl of H2O + 2.3µl of 1mM Actinomycin D} should have A441nm  =  0.5
» add more H2O to ajust concentration to 1M

  • Working solution, 20x (~0.1M, 120 ng/µl): add it to the First-strand synthesis reaction just before adding FS enz. mix

  1. for processing of several samples in parallel prepare 3.3xFS mixture for all samples
  2. temperature changes slowly to increase reproducibility of the reaction.
  3. PCR machine:
    • steps 1-2: RNA denaturation;
    • steps 3-4: primer annealing;
    • step 5: enzyme loading.
  4. add enzymes by 2µl pipette; mix using the same tip, but 20µl pipette.
  5. PCR machine:
    • steps 6-11: cDNA synthesis at gradually increasing temperature;
    • step 12: enzyme inactivation.
  6. buffer should be changed before use
  7. for processing of several samples in parallel prepare 3.3xSS mixture for all samples
  8. alternative to QIAquick: phenol-chloroform extraction and ethanol precipitation;

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