RNA extraction from cells cultured on ACCs

We have tried to perform RNA extraction from MSC growing and differentiating cells using several protocols. The protocol described here is the adaptation from the manufacturer’s instructions with which we were able to successfully extract RNA from MSC cells growing and differentiating on a carbon cloth matrix.

Commercial kit used: Purelink RNA Mini Kit (Life Technologies, 12183018A). Manufacturer’s full protocol is available on their webpage.

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Reverse transcription of RNA samples into cDNA

If we want to study gene expression we must do a reverse transcription to convert mRNA from our total RNA samples into cDNA before performing qPCR. By doing this we’ll be able to amplify expressing genes, and as our cDNA templates lacks introns, we could design primers that only amplifies cDNA, avoiding this way the amplification of gDNA contaminants or if it is not possible, design primers separated by at least one intron to discriminate between cDNA-amplified products from gDNA-amplified ones.

This protocol is optimized to fit our needs from the original manufacturer’s protocol. We are using a commercial from Bio-rad (iScript™ cDNA Synthesis Kit). Manufacturer’s full protocol is available on their webpage.

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