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Quantification of Sirt1 transcripts in rat brain tissue (cDNA qPCR)

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Quantify Sirt1 relative to GAPDH in cDNA prepared from rat brain RNA using separate SYBR Green qPCR reactions.

Scope and method

Sample：cDNA obtained by reverse transcription of rat brain tissue RNA; 10 ng cDNA per well.

Real-time quantitative PCR (qPCR)

Bill of materials (BOM)

StepMaterialRoleAmountRequired / optionalSubstitution conditionsSKU / specificationLiterature product
Addition of cDNALightCycler 480 384-well reaction plate (Roche 4729749001)Used for Sirt1 and GAPDH qPCR reactions on the LightCycler 480.

The source specifies Roche Diagnostics 4729749001 with 384 wells. This is a source material reference, not a company sales SKU or supply commitment.

Not reported in the sourceRequiredNot reported in sourceAvailable for inquiryNot reported in source
Preparation of reaction premixPowerUp SYBR Green Master Mix（A25776）SYBR Green PCR Master Mix

PowerUp SYBR Green Master Mix（A25776）。

5 µL/reaction wellRequiredNot reported in sourceAvailable for inquiryNot reported in source
Preparation of reaction premixRat Sirt1 qPCR forward and reverse primer pairSirt1 target gene primers

Use the listed rat Sirt1 forward and reverse primer sequences; separate from the GAPDH reaction.

Add 1 µL of forward and reverse primer working mix (5 µM) to the Sirt1 reaction well.RequiredNot reported in sourceAvailable for inquiryNot reported in source
Preparation of reaction premixRat GAPDH qPCR forward and reverse primer pairGAPDH internal reference primers

Use the listed rat GAPDH forward and reverse primer sequences; separate from the Sirt1 reaction.

Add 1 µL of forward and reverse primer working mix (5 µM) to the GAPDH reaction wellsRequiredNot reported in sourceAvailable for inquiryNot reported in source
Preparation of reaction premixNuclease-free waterWater for primer dilution

Nuclease-free water, suitable for qPCR.

Used to prepare a 5 µM primer working mixture; the amount of water to add is determined by the concentration of the primer stock solutionRequiredNot 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 #185 · revision 3 LightCycler 480 384-well reaction plate (Roche 4729749001) | Not reported in the source | Available for inquiry PowerUp SYBR Green Master Mix（A25776） | 5 µL/reaction well | Available for inquiry Rat Sirt1 qPCR forward and reverse primer pair | Add 1 µL of forward and reverse primer working mix (5 µM) to the Sirt1 reaction well. | Available for inquiry Rat GAPDH qPCR forward and reverse primer pair | Add 1 µL of forward and reverse primer working mix (5 µM) to the GAPDH reaction wells | Available for inquiry Nuclease-free water | Used to prepare a 5 µM primer working mixture; the amount of water to add is determined by the concentration of the primer stock solution | Available for inquiry

Procedure

  1. SYBR Green reaction

    SYBR Green qPCR。

  2. Preparation of reaction premix

    For each well, prepare 5ul PowerUp SYBR Green Master Mix (A25776, Applied Biosystems–Thermo Fisher) and 1ul working mix of forward and reverse primers (diluted to 5 µM in nuclease-free water).

  3. Dispensing the premix

    Use a Multipette E3 automatic pipette to dispense 6ul SYBR premix into each well.

  4. Addition of cDNA

    Add 4ul cDNA (10 ng) to each well of a LightCycler 480 384-well reaction plate (Roche, 4729749001); set up 3 technical replicates for each assay.

  5. Real-time amplification

    Use the LightCycler 480: 50˚C for 2 min; 95˚C for 2 min; then 95˚C for 15 s and 60˚C for 1 min, repeated for 40 cycles.

  6. Reading Ct values

    Obtain the threshold cycle (Ct) for each sample using LightCycler software.

  7. Expression analysis

    Analyze expression data by the ΔΔCt method using the reference gene and the control experimental group.

Quality control and readouts

Check the Sirt1 and GAPDH primer sequences and assess amplification specificity, technical replicate consistency, and negative controls separately. Use consistent sample handling and analysis across comparison groups. Technical replicates do not count as independent animals. Confirm that GAPDH expression is stable under the study conditions before using it as a reference gene.

Ct values for Sirt1 and GAPDH, ΔCt, ΔΔCt relative to the control group, and relative Sirt1 expression (2^−ΔΔCt).

Study design
Set up separate Sirt1 and GAPDH reactions, with 3 technical replicates per assay for each sample; compare normalized expression between experimental and control groups.

References

  1. Vila M. Gene expression analysis by quantitative Real-Time PCR (qPCR). protocols.io, v1, 2022. DOI:10.17504/protocols.io.ewov1oyz2lr2/v1. · DOI: 10.17504/protocols.io.ewov1oyz2lr2/v1
  2. Schisandrin ameliorates cognitive deficits, endoplasmic reticulum stress and neuroinflammation in streptozotocin (STZ)-induced Alzheimer’s disease rats. Experimental Animals. PMID:32336744. DOI:10.1538/expanim.19-0146. · DOI: 10.1538/expanim.19-0146
  3. Bustin SA, et al. MIQE précis: Practical implementation of minimum standard guidelines for quantitative real-time PCR experiments. BMC Molecular Biology 2010;11:74. PMID 20858237. doi:10.1186/1471-2199-11-74 · DOI: 10.1186/1471-2199-11-74

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