Pre-PCR RNA Amplification
When working with very small samples, whether limited amounts of sorted cells, laser capture microscopy samples or small tissues samples, obtaining sufficient RNA to perform a series of qRT-PCR assays can be a problem. We have validated a method to perform a linear amplification on small RNA samples to allow for archiving and qRT-PCR. Amplification uses a random primed, single primer isothermal amplification method (SPIA) developed by NuGEN Technologies.
In the example below, total RNA was obtained from the entire mouse hippocampus or dissected dentate gyrus regions from four to five 450μm thick horizontal brain slices. 10 ng RNA aliquots from the dentate gyrus were SPIA amplified, generating 1-2 μg of amplified cDNA product. In comparison, standard reverse transcription (RT) reactions (Applied Biosystems, Foster City, CA) were performed using 300 ng of DNase-treated hippocampal total RNA with random hexamer and oligo-d(T) primers (2.5 μM each).
Standard curves were made with serial dilutions of pooled cDNAs (30, 6, and 1.2 ng) from the SPIA amplified and standard RT reactions. The resulting cDNAs were assayed by qPCR in parallel with SYBR green PCR reagents in duplicate on an ABI Prism 7700 sequence detector (Applied Biosystems). Three representative samples, amplified and unamplified, gave indistinguishable amplification profiles in our hands (shown below).
