Recovering RNA From Small Samples RNAqueous™-Micro Kit
Molecular analysis of gene expression from very small samples can be challenging. The RNA isolation method used for limited samples should provide for quantitative recovery of RNA that is intact, free from contaminants, and as highly concentrated as possible. Increasingly, expression analysis is being carried out on "micro-sized" samples, including samples obtained by Laser Capture Microdissection (LCM) and related microdissection techniques; needle biopsies; fine dissection; and samples comprised of low numbers of cultured cells. In these cases, it is desirable to recover the total RNA in a small volume so that the entire sample can be used in downstream applications, such as reverse transcription. Also, keeping the sample as concentrated as possible facilitates analysis and quantitation of the total RNA using sensitive microfluidic assays (e.g. Agilent 2100 bioanalyzer). Ambion's RNAqueous™-Micro Kit was developed to optimize recovery of highly concentrated total RNA from micro-sized samples.
Isolate Exceedingly Pure Total RNA in a Small Volume
High Quality RNA for qRT-PCR
Figure 2 shows an electropherogram and real-time PCR data generated from cells isolated by LCM and RNA purified using the RNAqueous-Micro Kit. The RNAqueous-Micro Kit contains sufficient reagents and filter cartridges to isolate total RNA free of DNA from 50 samples of 1 - 1 x 105 cells or up to 5 mg of tissue.
Figure 1. (A) Linear Recovery of RNA from Cells Over a 5 Log Range. RNAqueous™-Micro Kit was used to isolate total RNA from K562 cells diluted from 100,000 cell equivalents down to 1 cell equivalent. The RNA samples were then used for amplification of GAPDH by real time RT-PCR. (B)Electropherogram of RNA isolated from 10,000 K562 cells. RNA was run on an Agilent 2100 bioanalyzer. Total yield after DNase treatment and clean up was 360 ng with an 28S/18S rRNA ratio of 1.87.
Figure 2. (A) Use of LCM Samples for One Step RT-PCR of Neurogranin. RNA was isolated from nine mouse brain Laser Capture Microdissection samples using the RNAqueous™-Micro Kit. The 20 µl post isolation eluates were DNase treated (final volume 25 µl). A fifth of this treated sample was subjected to one-step RT-PCR for analysis of neurogranin (Ct of 18.44).
(B) Electropherogram of RNA Isolated from the Granule Cell Layer of Hippocampus (Mouse Brain). Cells were obtained through LCM and RNA was isolated with the RNAqueous™-Micro Kit. 5 µl was run and analyzed on an Agilent 2100 bioanalyzer. Total yield after DNase treatment and clean up was 120 ng with an 28S/18S rRNA ratio of 0.72.