How can a convenient result about miRNA be obtained by RT-PCR from FFPE breast tissues?

More Sevan Majed's questions See All

Can somebody interpret the following images belonged to a miRNA pretest?

Dear/Madam I am a Ph.D. student. I am working on microRNA expression in FFPE breast tissue. Today I did a pretest for one sample (Cancer) to detect one microRNA. I ran a breast cancer sample and...

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Can I synthesize single strand cDNA for miRNA from total RNA? or Can I use Total RNA extracted to isolate microRNA?

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Does anybody has the protocol of MicroRNA purification from FFPE block tissue?

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Can I use DNase I during washing microRNA Enrichment Column?

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Arnaud Beurdeley

Dear Sevan,

miRNAs purification from FFPE should be tricky sometimes. Could you please gave few details on your workflow... I will ask you few questions/recommendations below:

Did you use a dedicated kit to enrich your extracts in miRNAs (I would not recommend it) or use a total RNA purification kit with a specific work flow to sort miRNAs?

Did you use glycogen as RNA carrier? this will improve your RNA-recovery yield.

Did you quantify/qualify your miRNAs extractions? or as mainly described use a specific volume of RNA to run your RT? Is it possible to check for RNA purity (absorbance ratio). if Absorbance A260/A280 is to low it might interfere with the first step of your RT for miRNas : ligation of an oligo to increase the size of your miRNAs.

Have you got some positive controls in your work flow (spike in during extraction, spike for RT and qPCR positive controls?).This will help you to check which step of your WF goes wrong....

What is your elution volume? in what buffer? I would recommend TE1x or TE0,5x instead of DEPC or RNAse Free water

Regarding the RT, I will recommend to work with universal priming instead of primer specific (RT from life technologies for taqman advanced assays). If you prefer using Sybr Green during qPCR, I would recommend to shift to Exiqon products (Qiagen).

I guess that's a lot of point for you, but If needed do not hesitate to contact me with your protocol is possible, I will be pleased to help.

Best.

Arnaud

Sevan Majed

Dear Arnaud Beurdeley

Many thanks that you have provided a lot of recommendation.

1. I have used Norgen kits for miRNA purification.

2. I have not used glycogen because it does not mention in the procedure. If you have any recommendation about using glycogen please tell me, I will be grateful.

3. Regarding absorbance A260/A280, One of my sample is about 13.41.

4. That is a good point. I have not positive control. I will be used it.

5. I have used SyberGreen during qPCR.

Please the attached file that is my procedure

Valeria De Arcangelis

Hi, I don't have experience with FFPE tissue but I know the yield of the RNA is low and the RNA is easily degraded, in addition you need enrichment in small RNAs.

I known that you use other kit company but Qiagen provide some brochure with useful tips. It is about possible troubleshooting with FFPE sample. Naturally they promote their product but you can ignore this part.

https://www.qiagen.com/lu/resources/resourcedetail?id=00d71edd-0bc1-4901-8642-4b55bd0ed47a&lang=en

For the final real time step, I would consider switch to Taqman and add a preamplification step it is a very important with low yield RNA samples.