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cDNA Concentration PCR

Goals

  • Test that primers work
  • Verify single products via a melt curve analysis
  • Determine what dilution of your cDNA you will use as template in subsequent reactions
  • Test RNA for gDNA contamination

Typical plate setup for single reference gene and single target (you must test all candidate reference genes):

1

2

3

4

5

6

A

5

5

5

5

5

5

B

25

25

25

25

25

25

C

125

125

125

125

125

125

D

625

625

625

625

625

625

E

3125

3125

3125

3125

3125

3125

F

15625

15625

15625

15625

15625

15625

G

RNA

RNA

RNA

RNA

RNA

RNA

H

50nM

50nM

200nM

200nM

500nM

500nM

Ref

Target

Numbers are cDNA dilution, Concentrations are [primer]

Conditions

  • The cDNA used for this is a pool of all your cDNA samples i.e. take 1ul of each RT reaction product, pool them, and make dilutions from there. Why? You don't know the difference in expression between your experimental and control sample, or the variation in your biological replicates. If you just ran this plate using cDNA template from one reaction, you might end up choosing conditions suitable for that sample, but not suitable for the others. This limits that error.
  • The RNA sample used to test for gDNA contamination is a pool of all your RNA samples (just like you did for the cDNA pool in the step above). This pool is then diluted to the same extent as the 1:25 cDNA dilution i.e. take Xng of your pooled RNA (x = ng you inputted into RT), make up to 20ul (because your initial reverse transcription was 20ul), and then make a 1:25 dilution.

What you are looking for

  • PCR products that produce a single peak in your melt curve analysis
  • A dilution of cDNA that produces Cq values of between 13-30 cycles (If your Cqs are less than or greater than this, the assay can still work, but depending on machine, standard deviations can get a bit noisy if amplification happens too early or late.)
  • No contamination in RNA, or contamination that is >5 cycles later than the signal in your experimental sample. For primers that are 100% efficient, each cycle represents a doubling of product. Therefore a difference of 7 cycles would be a difference in expression of 2^7 or 128 fold.

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Topic revision: r2 - 21 Mar 2019 - 21:46:43 - Main.KateElston
 
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