Single cell RNA and mutational analysis PCR (SCRM-PCR): a method for simultaneous analysis of DNA and RNA at the single-cell level

A method of simultaneously analyzing RNA and DNA in a sample, the method comprising the operations of:

• (a) contacting the sample with a reverse primer from a first primer pair, the reverse primer from the first primer pair being directed to a target RNA region, and a reverse transcriptase to effect reverse transcription of the RNA into cDNA;
• (b) subsequently contacting the sample with:
• (i) a forward primer from the first primer pair, the forward primer from the first primer pair being directed to a target cDNA region,
• (ii) a reverse primer and a forward primer from a second primer pair, the reverse primer and forward primer from the second primer pair being directed to a target DNA region, and
• (iii) a DNA polymerase to simultaneously amplify the target cDNA region and the target DNA region;
• (c) (A) subjecting the sample from operation (b) to a semi-nested PCR using the reverse primer in operation (a) or the forward primer in operation (b)(i), a nested primer that binds within the pre-amplified target cDNA region, and a DNA polymerase to further amplify the target cDNA region;
• and/or
• (B) subjecting the sample from operation (b) to a nested PCR using a nested primer pair that binds within the pre-amplified target DNA region and a DNA polymerase to further amplify the target DNA region;
• and
• (d) analyzing the amplified target cDNA region and/or the amplified target DNA region;
• wherein the sample comprises a single cell;
• and
• wherein prior to operation (a), the method does not comprise lysing the cell in the sample.