Skip to main content

First-strand cDNA synthesis from brain tissue RNA

Browse experimental protocols

Synthesize first-strand cDNA from purified brain total RNA using ProtoScript II reverse transcriptase.

Scope and method

Sample：Purified total RNA from brain tissue.

Reverse transcription and cDNA synthesis

Bill of materials (BOM)

StepMaterialRoleAmountRequired / optionalSubstitution conditionsSKU / specificationLiterature product
Prepare the RNA and primer mixtureSterile microcentrifuge tube for cDNA synthesisPrepare the brain-tissue RNA, primer and dNTP mixture for first-strand cDNA synthesis.

The source explicitly specifies a sterile microcentrifuge tube but does not report tube capacity or the total required per experiment.

Not reported in the sourceRequiredNot reported in sourceAvailable for inquiryNot reported in source
Prepare the reverse transcription reactionProtoScript II Reverse Transcriptase（M0368S）First-strand cDNA synthesis

ProtoScript II Reverse Transcriptase，M0368S，200 U/µl。

1 µl/20 µl reactionRequiredNot reported in sourceAvailable for inquiryNot reported in source
Prepare the reverse transcription reaction5X ProtoScript II RT Reaction BufferReverse transcription buffer system

5X ProtoScript II RT Reaction Buffer。

4 µl/20 µl reactionRequiredNot reported in sourceAvailable for inquiryNot reported in source
Prepare the reverse transcription reactionMurine RNase Inhibitor（M0314S）RNase inhibition

Murine RNase Inhibitor，M0314S，40 U/µl。

1 µl/20 µl reactionRequiredNot reported in sourceAvailable for inquiryNot reported in source
Prepare the reverse transcription reaction0.1 M DTT reverse transcription working solutionReverse transcription reconstitution components

0.1 M DTT working solution.

2 µl/20 µl reactionRequiredNot reported in sourceAvailable for inquiryNot reported in source
Prepare the RNA and primer mixturedNTP Mix (N0447S)cDNA synthesis substrate

dNTP mix, N0447S; 10 mM working solution.

1 µl/20 µl reactionRequiredNot reported in sourceAvailable for inquiryNot reported in source
Prepare the RNA and primer mixtureReverse transcription random primer mix (60 µM)Reverse transcription primer

60 µM Random Primer Mix; when using random primers, retain the 25 °C annealing step.

2 µl/20 µl reactionRequiredNot reported in sourceAvailable for inquiryNot reported in source
Prepare the RNA and primer mixtureNuclease-free waterReactive hydration

Nuclease-free water.

After adding RNA, primers, and dNTPs, make up the volume to 12 µl.RequiredNot reported in sourceAvailable for inquiryNot reported in source
Inquire about the whole BOM

Bulk pricing and availability inquiry

Submitting an inquiry does not place an order or confirm a purchase.

Protocol #187 · revision 3 Sterile microcentrifuge tube for cDNA synthesis | Not reported in the source | Available for inquiry ProtoScript II Reverse Transcriptase（M0368S） | 1 µl/20 µl reaction | Available for inquiry 5X ProtoScript II RT Reaction Buffer | 4 µl/20 µl reaction | Available for inquiry Murine RNase Inhibitor（M0314S） | 1 µl/20 µl reaction | Available for inquiry 0.1 M DTT reverse transcription working solution | 2 µl/20 µl reaction | Available for inquiry dNTP Mix (N0447S) | 1 µl/20 µl reaction | Available for inquiry Reverse transcription random primer mix (60 µM) | 2 µl/20 µl reaction | Available for inquiry Nuclease-free water | After adding RNA, primers, and dNTPs, make up the volume to 12 µl. | Available for inquiry

Procedure

  1. Prepare the RNA and primer mixture

    In a sterile centrifuge tube, combine RNA (1ng-1 µg total RNA or 1-100 ng poly(A) RNA), 2 µl primer (50 µM dT or 60 µM Random Primer Mix), and 1 µl 10 mM dNTP mix. Bring to 12 µl with nuclease-free water.

  2. RNA denaturation

    The mixture may be heated at 65 °C - 70 °C for 3-5 minutes, briefly centrifuged, and immediately placed on ice.

  3. Prepare the reverse transcription reaction

    Add 4 µl 5X ProtoScript II RT Reaction buffer, 2 µl 0.1 M DTT, 1 µl Murine RNase inhibitor (40 U/µl), and 1 µl ProtoScript II Reverse Transcriptase (200 U/µl), giving a total volume of 20 µl.

  4. Random-primer annealing

    When using Random Primer Mix, incubate at 25 °C for 00:05:00 before the reaction at 42 °C.

  5. Reverse transcription

    Incubate at 42 °C for 30 minutes-1 hour.

  6. Enzyme inactivation

    Incubate at 80 °C for 00:05:00.

  7. Store cDNA

    Store the product at -20 °C or proceed to the subsequent assay.

Quality control and readouts

Use intact RNA free of RNase contamination; record the input amount for each sample and check no-reverse-transcription controls. Use consistent cDNA input amounts for subsequent quantification.

First-strand cDNA for subsequent amplification of target sequences.

References

  1. New England Biolabs. First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368), protocols.io v2, 2022-04-11. · DOI: 10.17504/protocols.io.x14egnmmv5dy/v2
  2. Schisandrin ameliorates cognitive deficits, endoplasmic reticulum stress and neuroinflammation in streptozotocin (STZ)-induced Alzheimer’s disease rats. PMID:32336744. · DOI: 10.1538/expanim.19-0146

Technical services

    Browse technical services