Generating microRNA standard curve

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Generating microRNA standard curve

Postby jdenk » Wed Aug 26, 2015 1:00 pm

Dear PCR community,

i'm doing relative "microRNA" expression exeriments using the Exiqon LNA System.

However, i'd like to generate a standard curve using a synthetic miRNA to address the following (MIQE conform) analytical parameters to see how good my protocol works:

- Sensitivity (LOD function in GenEx), (this is important to have an idea about the Cq cut-off of your system when analyzing the data later on),
- intra-assay variance (technical PCR repeats),
- linar dynamic range,
- PCR efficiency (at least for the analyze spike),
- specificity (NTC, melting curves).

All these parameters should be covered by generating proper designed standard curve(s). However, there are several recommendations (doi:10.1016/j.bdq.2015.01.005) and different possibilities for standard curves. I appreciate any helpful answers to the following points:

1. Should the serial dilution be based on "total RNA" (spike added during isolation), "cDNA" (spike added prior cDNA synthesis), or diluted spikes (which are added prior cDNA syntheses but after they were diluted)?
2. how many logs to cover? (i'm rather interested in the Cq variation at low concentrations)
3. which transfer volume to use during serial dilution?
4. which dilution factors: 1:2, 1:5, 1:10 ?
5. how many RT and/or PCR replicates needed (at least)?

I'm going to use a 96-well plate - so I try to set up the expriment in a way that I can avoid inter-run variation - otherwise I would need IPCs. The experimental design should give results that I can use to publish the data as MIQE conform.

I'm thankful for any recomendations!!!
jdenk
 
Posts: 11
Joined: Fri Feb 22, 2013 11:27 am

Re: Generating microRNA standard curve

Postby Mikael Kubista » Tue Apr 26, 2016 10:53 am

1. Should the serial dilution be based on "total RNA" (spike added during isolation), "cDNA" (spike added prior cDNA synthesis), or diluted spikes (which are added prior cDNA syntheses but after they were diluted)?

The total amount of RNA should be the same for all samples, to avoid non-linearity:
Anders Ståhlberg, Joakim Håkansson, Xiaojie Xian, Henrik Semb, and Mikael Kubista
Properties of the Reverse Transcription Reaction in mRNA Quantification
Clinical Chemistry 50:3, 509-515, 2004

2. how many logs to cover? (i'm rather interested in the Cq variation at low concentrations)

The larger range covered, the more precise will be the estimated parameters. Use GenEx (www.tataa.com) to obtain the confidence intervals and you will see how the precision improves.

3. which transfer volume to use during serial dilution?

The larger the better, since it will reduce pipetting errors, which usually is a dominant source of error. Never pipette less than 1 ul.

4. which dilution factors: 1:2, 1:5, 1:10 ?

The larger concentration range spanned the better. Small dilution steps usually only cover limited range.

5. how many RT and/or PCR replicates needed (at least)? Depends on reproducible those steps are in your assay. Technical replicates shall be averaged prior to analysis. Only samples count as different standard points!

David Svec , Ales Tichopad, Vendula Novosadova, Michael W. Pfaffl, Mikael Kubista
How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments
Biomolecular Detection and Quantification (2015) doi:10.1016/j.bdq.2015.01.005

Cited publications are available on: www.tataa.com
Mikael Kubista
 
Posts: 455
Joined: Tue Jul 01, 2008 12:28 pm


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